EFFECTS OF ELEVATED CO2 ON GROWTH, DEVELOPMENT, NUTRIENT
CONCENTRATION AND INSECT PERFORMANCE OF PLANTS GROWN AT
SUB-OPTIMAL TEMPERATURE
Thesis
Presented in Partial Fulfillment of the Requirement for the Degree of Master of Science
in the Graduate School of The Ohio State University
By
Wilmer Mauricio Rodríguez, B.S.
Graduate Program in Horticulture and Crop Science
The Ohio State University
2011
Thesis Committee:
Dr. Claudio Pasian, Advisor
Dr. Luis Cañas
Dr. Peter Ling
Dr. Jonathan Frantz
Copyright by
Wilmer Mauricio Rodríguez
2011
Abstract
The effect of elevated CO2 on growth, development, nutrient concentration and
insect performance was evaluated in petunias (Petunia×hybrida Vilm.) and zinnias
(Zinnia elegans L.) grown at sub-optimal air temperature. Elevated (700 µmol·mol-1) and
ambient (390 µmol·mol-1) CO2 concentrations were used to grow plants in chambers and
greenhouse environments using a recommended “optimal” (21 ˚C) and a sub-optimal (3
˚C lower than optimal) air temperature. Controlled Release Fertilizer (CRF) was used at
low and high rates to test the effect of CO2 and temperature on plant nutrition. Whiteflies
(Bemisia tabaci Gen.) were infested on plants to evaluate oviposition and nymph
development. The growth chamber study revealed that petunias and zinnias grown at
elevated CO2 were shorter than plants at low CO2 levels. High fertility increased plant
dry weight for both crops. While the number of flowers in zinnias was increased with
high fertility, flower longevity and time to flower were not affected. On the other hand,
higher number of flowers, longer flower longevity and shorter time to flower was
observed in petunias grown at high CO2 and sub-optimal temperature. N, K, Ca, Mg, S
and C were not affected by either CO2 or temperature treatments, whereas P had higher
concentrations in petunias grown at low CO2 with optimal temperature. However, in
zinnias, Mg was the only nutrient affected by CO2 levels. In the greenhouse study, higher
plant dry weights and heights were measured with high fertility in petunias but not in
zinnias. The number of flowers increased for both crops while the longevity and time to
ii
flower decreased for petunias and remained unchanged for zinnias with the high fertility
treatment. Nutrient concentrations in petunias were not affected by the CO2/temperature
combination whereas in zinnias an increase in C/N ratio was observed. While early
mortality of whiteflies infested on petunias caused insufficient data to evaluate insect
performance, zinnia‟s growth was negatively affected by the presence of insects.
Whiteflies‟ oviposition rate was reduced and nymph developmental time increased by
sub-optimal temperature at both chamber and greenhouse studies. It has been
demonstrated with this study that the combination of CO2 enrichment and a slight
reduction in air temperature may not compromise plant quality and scheduling.
Furthermore, positive results in terms of pest management and fertilization can be
achieved.
iii
Dedicated to my parents Lilian Rodríguez Portillo and Julio Cesar Interiano
iv
Acknowledgments
Thank you to my advisor Claudio Pasian for all the intellectual input and the
valuable mentorship, your criticisms and suggestions guided me to complete such an
important achievement in my life.
Many thanks to my committee members Luis Cañas, Peter Ling and Jonathan
Frantz for your wisely advice and patience. All your help and toughs helped me to
accomplish my goal and were essential to get me successfully through this program.
I certainly owe many thanks to Luis Cañas for giving me the initial opportunity to
do an internship at his laboratory and also for all his support and friendship throughout
this endeavor.
Thanks to all my friends, your love and support were so essential. I would not
have accomplished many of my goals without your help.
Thanks to all the personnel at the Insect Ecology and Controlled laboratory;
Ronald Batallas, Nuris Acosta, Karla Medina, Claudia Kuniyoshi, Jim Hacker and Mark
Belcher; your help was fundamental for the completion of this research. Definitely,
without your support this journey would have been almost impossible. I also want to
thanks Luke Power and the personnel at the nutrient analysis laboratory at Toledo
University for their help in this project.
v
Vita
Oct 15, 1983……Born - Ocotepeque, Honduras
2001…………….Technician in agriculture, School of Agriculture Pompilio
Ortega, Santa Barbara, Honduras
2005…………….B.S.
Agriculture,
Panamerican
School
of
Agriculture,
Zamorano, Honduras
2008 – Present….Graduate Research Associate, The Ohio State University
Field of Study
Major Field: Horticulture and Crop Science
vi
Table of Contents
Abstract ............................................................................................................................... ii
Dedication .......................................................................................................................... iv
Acknowledgments............................................................................................................... v
Vita..................................................................................................................................... vi
List of Tables ................................................................................................................... viii
List of Figures .................................................................................................................... ix
CHAPTER 1 ....................................................................................................................... 1
Introduction ..................................................................................................................... 1
Literature Review ............................................................................................................ 3
CHAPTER 2: Growth Chamber Study ............................................................................. 10
Introduction ................................................................................................................... 10
Materials and Methods .................................................................................................. 12
Results ........................................................................................................................... 21
Discussion ..................................................................................................................... 38
Conclusion..................................................................................................................... 47
CHAPTER 3: Greenhouse Study ...................................................................................... 49
Introduction ................................................................................................................... 49
Materials and Methods .................................................................................................. 51
Results ........................................................................................................................... 54
Discussion ..................................................................................................................... 67
Conclusion..................................................................................................................... 77
REFERENCES ................................................................................................................. 80
APPENDIX A: Environmental settings and protocol to rear whiteflies.......................... 87
vii
List of Tables
Table 1. Concentration of macronutrients, carbon and C/N ratios in the leaves of petunias
grown in growth chambers............................................................................28
Table 2. Concentration of macronutrients, carbon and C/N ratios in the leaves of zinnias
grown in growth chambers............................................................................34
Table 3. Concentration of macronutrients, carbon and C/N ratios in the leaves of petunias
grown in a greenhouse environment .............................................................59
Table 4. Concentration of macronutrients, carbon and C/N ratios in the leaves of zinnias
grown in a greenhouse environment. ............................................................64
viii
List of Figures
Fig.1.
Fig.2.
Fig.3.
Fig.4.
Fig.5.
Fig.6.
Fig.7.
Fig.8.
Fig.9.
Fig.10.
Fig.11.
Fig.12.
Fig.13.
Fig.14.
Fig.15.
Fig.16.
Fig.17.
Fig.18.
Fig.19.
Fig.20.
Fig.21.
Fig.22.
Clip cage to confine whiteflies is attached to a petunia leaf .........................19
First instar whiteflies circled in a petunia leaf. The development of the
nymph in each circled was followed until emergence ..................................20
Effects of temperature, CO2 and fertilization levels on zinnias‟s height. .....22
Effects of temperature and CO2 concentration on petunia‟s height.. ............23
Effects of CO2 and fertilization level on petunia‟s height.. ..........................23
Effects of temperature and fertilization level on the total number of flowers
in petunias. ....................................................................................................24
Effects of temperature, CO2 and insect conditions on the time to first open
flower in petunias.. ........................................................................................24
Effects of temperature and fertilization level on the time to first open flower
in petunias.. ...................................................................................................25
Effects of temperature on the average longevity of flowers in petunias. ......25
Effects of CO2 on the average longevity of flowers in petunias. ..................26
Effects of fertilization levels on zinnias‟s dry weight.. ................................30
Effect of insects on zinnia‟s dry weight.. ......................................................30
Effects of temperature, CO2 and fertilization levels on zinnias‟s height. .....31
Effects of fertilization levels on the total number of flowers in zinnias. ......31
Effects of temperature, CO2 and fertilization levels on the carbon-nitrogen
ratio of zinnias...............................................................................................33
Effects of temperature on oviposition rate (eggs per female per day) of
whiteflies. ......................................................................................................35
Effects of temperature and CO2 levels on nymph mortality of whiteflies
infested on zinnias.........................................................................................36
Effects of temperature on the degree days to the first whitefly emerged in
zinnias. ..........................................................................................................36
Effects of temperature on the degree days to the 50% whitefly emergence in
zinnias. ..........................................................................................................37
Effects of temperature on the degree days to 100% whitefly emergence in
zinnias. ..........................................................................................................37
Effects of fertilization levels on petunias‟s dry weight. ...............................55
Effects of fertilization levels on petunias‟s height. .......................................55
ix
Fig.23.
Fig.24.
Fig.25.
Fig.26.
Fig.27.
Fig.28.
Fig.29.
Fig.30.
Fig.31.
Fig.32.
Fig.33.
Effects of CO2/temperature combination on the total number of flowers of
petunias. ........................................................................................................56
Effects of fertilization levels on the total number of flowers of petunias. ....56
Effects of CO2/temperature combination on the average longevity of flowers
of petunias. ....................................................................................................57
Effects of CO2/temperature combination on the time to first open flower of
petunias. ........................................................................................................57
Effects of CO2/temperature combination and insect condition on zinnia‟s dry
weight. ...........................................................................................................61
Effects of insect on zinnia‟s height. ..............................................................61
Effects of CO2/temperature combination, insect condition and fertilization
level on the total number of flowers in zinnia. .............................................62
Effects of CO2/temperature combination, insect condition and fertilization
level on the carbon-nitrogen ratio of zinnias. ...............................................63
Effects of CO2/temperature combination on oviposition rate (eggs per
female per day) of whiteflies infested in zinnias. .........................................65
Effects of CO2/temperature combination on nymph mortality of whiteflies
infested in zinnias. ........................................................................................65
Effects of CO2/temperature combination on the degree days to 100%
whiteflies emergence in zinnias. ...................................................................66
x
CHAPTER 1
INTRODUCTION
Energy efficiency is one of the major challenges that the floriculture industry is
facing. Dieleman et al. (2006) indicated that energy efficiency can be improved by either
a reduction in energy consumption or an increase in crop productivity. Furthermore,
Dieleman et al.( 2006) also reported a reduction of 16% in energy consumption by
lowering the temperature set point by 2 ˚C. According to the last report of the Department
of Energy (DOE, 2007) the total energy used to heat greenhouses in the United States
accounts for US$ 214.3 million. The implementation of energy saving strategies depends
of the overall profitability of the operation (Elings et al., 2005), and the effect that new
techniques to save energy may have in plant growth, development, nutrition and plantherbivore interactions (Korner and Challa, 2003; Mercier et al., 1988).
Different approaches have been used to reduce energy consumption in greenhouse
production; however, few studies have focused specifically on the control of greenhouse
heating to decrease such consumption (Bailey and Seginer, 1989). Approaches such as
temperature integration (Dekoning, 1990; Elings et al., 2006), increase of relative
humidity (Elings et al., 2006) and use of different construction materials (Bakker, 2006)
have been used to reduce heating cost in greenhouse production. Furthermore, CO2
1
enrichment and/or temperature reduction have been suggested to accomplish such
reduction (Elings et al., 2005). Energy conservations of 20-25% can be accomplished by
reducing temperature set points, increasing humidity, using energy screens and allowing
fluctuations in the greenhouse climate (Dieleman et al., 2006).
To reduce heating usage, most approaches have incorporated changes in
environmental settings only. Consequently, an understanding of the effects that
temperature, CO2, fertilization rates, and insect presence can have in the physiological
processes of the plant needs to be investigated in more detail. If changes in environmental
settings are profitable, knowledge on their effect at the plant level needs to be provided to
growers (Dieleman et al., 2006). The challenge is that plants will respond differently to
such environmental changes, for example the response to CO2 has been demonstrated to
be different from specie to specie (Bazzaz, 1990; Reekie et al., 1997).
2
LITERATURE REVIEW
Effects of air temperature on plant growth and development. Air temperature
more than any other environmental factor, affects plant growth and development,
especially the reproductive processes that are important in determining crop yield and
quality (Thuzar et al., 2010). While plant growth is defined as an increase in size, not
only in volume but also in height, plant development is defined as the differentiation of
cells into tissues, organs and organisms (Salisbury and Ross, 1992). Plant growth can be
measured by an increase in plant volume or an increase in plant weight (Salisbury and
Ross, 1992). On the other hand, measurement of plant development is more complicated
since there is a problem in providing detailed information of the description of the timing
and physiological sequence of the developmental events. However, factors such as the
control of number of flowers initiated, flowering times, fruit and seed development are
considered in the developmental phase of the plants (Johnson, 1981; Levitt, 1969).
A reduction in air temperature can cause a delay in plant growth and development
as the rate of chemical reactions (e.g. photosynthesis and respiration) decreases (Reid,
1991). Reducing the photosynthetic rate would most likely result in reduced growth
(Sage and Sharkey, 1987). In addition, studies focused on plant development support that
low temperatures can have negative effects on flowering time. Shimizu et al. (2002)
demonstrated that time to flower is longer in plants grown at low temperatures than those
grown at optimal or increased temperatures. However, Erwin et al. (1994) reported that
lower or higher than optimal temperatures can either inhibit, delay or reduce flowering
depending on the specie. Reductions in temperature have been tested in the United
Kingdom as an alternative to the use of growth regulators to prevent fast growth in
3
plants; results indicated that delay in plant growth and development negatively affected
the production scheduling, thereby increasing the time for plants to reach a marketable
size (Taylor et al., 2009).
Carbon Dioxide Enrichment. Elevated CO2 has been widely used to promote
plant growth and development (Bazzaz, 1990). Some studies have demonstrated that
faster and better production can be obtained at elevated CO2 with higher temperatures
(Sage et al., 1995), however little is known about the interaction of CO2 with lower than
optimal or recommended growing temperatures. Even though effects of high CO2 on
plants are variable, plants grown under enriched CO2 conditions often exhibit enhanced
photosynthetic activity and increased biomass and productivity (Coll and Hughes, 2008;
Hughes and Bazzaz, 1997; Owensby et al., 1999). Increased CO2 concentration enhances
the photosynthesis processes (Bazzaz, 1990; Li et al., 2007) affecting both growth and
phenology, especially in C3 plants (Reekie and Bazzaz, 1991). Such enhancement in
photosynthesis will drive faster plant growth and more biomass accumulation at maturity
(Moran and Jastrow, 2010; Wayne et al., 2002). For example, studies on Arabidopsis
thaliana have shown a gain in relative growth rate and leaf area with the greatest effects
at the early stages (Gibeaut et al., 2001). At high CO2 levels, a general plant response is
the accumulation of carbon in the form of starch and sugars (Poorter and Navas, 2003),
which may be reflected in a increase the total dry weight (Zhou and Shangguan, 2009).
Nevertheless, the degree to which plants can respond and acclimate to ambient or
elevated CO2 concentrations is probably influenced by environmental factors and time of
their availability (Bazzaz, 1990).
4
The effects of enriched CO2 conditions on plant development have also been
documented. Reekie et al. (1997) tested interactions between CO2 and daylengths finding
an increase in the number of flowers and buds as a result of exposure to elevated CO2.
Contrary to the increase obtained by Reekie et al. (1997), results obtained with Guara
brachycarpa showed that development was delayed when exposed to elevated CO2
concentrations, thus reducing time to flowering (Reekie and Bazzaz, 1991).
Other studies have focused on seed production to determine the effect of CO2 on
plant development; for instance, in four legumes (Glycine max, Phaseolus vulgaris,
Pisum sativum, and Vigna angularis) studied by Miyagi et al. (2007), significant increase
on seed production was observed. The effects of elevated CO2 on physiological processes
involving stomata conductance, photosynthetic enhancement and acclimation time have
been also demonstrated, which can also affect plant development (Piikki et al., 2007).
Elevated CO2, air temperature, and plant nutrition. A sustainable floriculture
production system not only requires an effort to decrease energy usage but also an
optimization of the fertilization program. Many authors hypothesize that an increase in
plant growth due to elevated CO2 concentrations will require a higher fertilization level
than the recommended level when growing plants at ambient CO2 (Li et al., 2007).
Goverde et al. (2002) demonstrated that responses on the quality of growth and tissues of
plants exposed to elevated CO2 are strongly affected by soil nutrient availability. They
also suggested that such responses will be accentuated with nutrient enriched conditions.
Hence, fertilization practices may have greater influence on the rate of nutrient
concentration at elevated than at ambient CO2 concentrations. Under elevated CO2 high
5
fertilization can lead to an increase in plant dry weight (Stiling and Cornelissen, 2007),
whereas at low fertilization the same increase in dry weight may not be achieved
(McMurtrie et al., 2008). In addition, it has been demonstrated that changes in carbon and
nitrogen dynamics may also be affected by CO2-temperature interaction (Himanen et al.,
2008).
Particularly, nitrogen has been the most studied nutrient at elevated CO2
concentrations. Reduction in growth has been observed when plants have limited N
availability, even at elevated CO2 conditions (McMurtrie et al., 2008). Several
researchers (Cotrufo et al., 1998; Curtis and Wang, 1998; Goverde et al., 2002; Korner
and Challa, 2003; Taub and Wang, 2008; Yin, 2002) have found that N concentrations
within the plant tissue are usually lower at high CO2 than at ambient levels. Taub and
Wang (2008) have provided a critical discussion to explain the reduction in N at elevated
CO2 concentrations. In their analysis they discuss the hypotheses of the dilution effect,
the decreased N uptake, the decreased N demand, the elevated CO2-mediated N loss, and
the ontogenetic drift in N concentration to provide a better understanding of the reduced
N at high CO2.
The information available regarding the effects of CO2 and temperature on
nutrient concentrations other than Nitrogen (N) is scarce. The physiological importance
of phosphorus and its role in plant metabolism is well understood; nevertheless,
understanding of the uptake by the plant when exposed to elevated CO2 levels is limited.
Suggestions have been made that P availability affected by elevated CO2 will be indirect
6
and mediated by the response of the biotic part of the ecosystem, which includes
geochemical and biological processes of the soil organic matter (Lukac et al., 2010). A
study carried out by Li et al. ( 2007) evaluating the P uptake of tomato seedlings exposed
at elevated CO2 concentrations demonstrated that P contents in roots, stems, and leaves
increased in the high-strength nutrient solution, whereas in the low strength solution the P
content decreased.
Similar results were obtained for K contents with the only difference that content
for stem tissues were variable depending of the nutrient solutions. Furthermore, in a
recent study evaluating how light, CO2 and fertility influence the growth, partitioning and
nutrient concentrations of Petunias cv. „Madness white‟; the researchers reported that
tissue concentrations of N, P and K were higher when fertility levels were increased from
7.1 to 23.3 mM N on those plants harvested three weeks after transplanting (Frantz and
Ling, 2011). On the same study of Frantz and Ling (2011), plants harvested at five weeks
after transplanting, only N was increased when fertility was increased, whereas in plants
harvested at seven weeks after transplanting high fertility increased concentrations of P
and K but decreased N tissue concentrations.
Elevated CO2, air temperature and insects. Pest management is also an
important aspect of the floriculture production. Knowledge of the insect response to
changes in the environmental variables can provide valuable information for pest
management and chemical applications. While it is well known that deleterious effects on
insect performance (oviposition rate, mortality, growth and developmental time) will be
observed at reduced temperatures, effects of CO2 enrichment have not been extensively
7
studied and the results available are inconsistent. Effects of temperature and CO2 on
insect performance can be direct by changing insects behavior or indirect by changing the
host nutritional quality resulting in changes of feeding behavior (Stiling and Cornelissen,
2007).
Whiteflies (Bemisia tabaci Gen.) are one of the most important pests in
floriculture crops; they can reduce the turgor pressure by tapping into the sap of the
plants. In large numbers the damage can be severe; moreover, whiteflies can transmit
geminivirus and also promote mold formation by the excretion of honeydew as their
waste product, which can affect the leaf photosynthetic rate (Byrne and Bellows, 1991).
Since insecticides accounted for 17.7 % of the 190,441 chemical applications in 2007 for
the six states evaluated in United States (USDA, 2007), success in controlling pests by
either reducing temperature or increasing CO2 may result in significant savings in
chemical applications.
Although it is well known that growth and development of whiteflies will be
negatively affected by lowering growing temperatures, few studies have been conducted
on evaluating the effect of elevated CO2 on these insects. Butler et al. (1983)
demonstrated that development, oviposition and longevity of whiteflies are closely
related to temperature. For instance, the developmental time from eggs to adults was
shown to be increased at low temperatures (Wang and Tsai, 1996). Furthermore, it has
been demonstrated that oviposition rate are affected by the environmental conditions and
the quality of the host plants; such variation can be from 8.3 to 39.6 eggs per female
(Byrne and Bellows, 1991).
8
Studies with other insects have been conducted under elevated CO2 as well.
Himanen et al. (2008) conducted an experiment with aphids (Misus persicae) and
demonstrated that high concentrations of CO2 significantly lowered the adult‟s weight
compared with aphids feeding from plants grown at ambient CO2. A decline of 21.6% in
leaf miner populations under elevated CO2 compared with ambient CO2 treatments was
also documented (Stiling and Cornelissen, 2007). Furthermore, increase in insect
consumption of leaves was detected when caterpillars were exposed to elevated CO2
(Schadler et al., 2007). Contrasting with some other positive effects of elevated CO2
mentioned before, (Coll and Hughes, 2008) found that whitefly females‟ oviposition or
adult longevity was not affected by elevated CO2.
Elevated CO2, plant nutrition and insects. It is clear that nutrition can affect
insect performance (Himanen et al., 2008). Insects will try to compensate by increasing
consumption when feeding from poor quality plants, which suggest the importance of
nutrient content within the plant (Herms and Mattson, 1992). In general, elevated CO2
has been found to change the plant quality (Marks and Lincoln, 1996; Schadler et al.,
2007) affecting the insect performance. Reduced N concentration in the plant (Mattson,
1980; Taub and Wang, 2008), increased leaf toughness at certain temperatures (Zvereva
and Kozlov, 2006), increased water concentration in the leaf (Bazzaz, 1990; Mattson,
1980), and increased content of carbon-based secondary metabolites such as phenolics or
alkaloids (Bidart-Bouzat and Imeh-Nathaniel, 2008) can negatively affect the
performance of herbivores, thereby affecting their feeding behavior, growth and
development.
9
CHAPTER 2
The effect of elevated CO2 on growth, development, nutrient concentration and
insect performance of plants grown in growth chambers at sub-optimal temperature
INTRODUCTION
Reproducible and specific environments for plant growth have been provided
through the traditional use of growth chambers (Fabreguettes et al., 1992). The advantage
of conducting studies in growth chambers is the precise and consistent control of
environmental settings throughout the duration of the experiment. A whole-canopy
approach can integrate several plant conditions which can include physiological stress,
entomological factors and also cultural practices such as fertilizations rates (Whiting and
Lang, 2001). Even though growth chambers provide a controlled environment care must
be taken to keep a good monitoring and recording system. The studies in chambers
provide a good control of environmental settings; therefore, we will use them to test all
the main effects and interactions of temperature, CO2 concentrations, fertilization levels
and insect effects on petunias and zinnias. In order to accomplish energy savings our
objective is to determine the effect of elevated CO2 on plant growth and development,
plant nutrient concentration, and insect performance when plants are grown at suboptimal temperatures. Based on the positive effects that elevated CO2 may have on plant
growth and development and taking into account the negative effects caused by lowering
10
temperature set point; we hypothesize that plant quality will be similar in those
plants grown at elevated CO2 and suboptimal temperature than in those grown at ambient
CO2 and optimal temperature. Moreover we are also expecting that insects will be
negatively affected by the sub-optimal temperature.
11
MATERIALS AND METHODS
Facilities. The chamber study was conducted in the Biosystem Phytotron facility
at the department of Food Agricultural and Biological Engineering at the Ohio
Agricultural Research and Development Center (OARDC), Wooster campus. The
phytotron is equipped with 12 plexiglas growth chambers 1 m3 each. The 12 chambers
are divided into two rows of six chambers. In one row, temperature, air flow and relative
humidity are controlled by an air handler (Model AA-5580A, Parameter Generation &
Control Inc. Black Mountain, NC), which supplies a continuous flow of conditioned air
over a range of 4.5 ºC to 71 ºC. The CO2 was injected into the chambers by tubing from
the main tank to the air handler and then dispersed to the six chambers in that row to
enrich their atmospheres.
Unlike the six chambers described previously, the other set of six chambers do not
have an air handler to pump conditioned air to control temperature and relative humidity.
The air flow was provided by a continuous pumping of outside air, which can result in
potential higher fluctuations than desired; thereby a more precise control of temperature
was needed. The incoming air is first blown through a heat exchanger to bring it closer to
room temperature before feeding into the chambers. Chamber air temperature is further
regulated using cooling coils that were installed along the inside wall of each chamber.
Water coming from a refrigerated circulating water chiller (6000 model, PolySience,
Niles, IL) was conducted into each of the coils to cool down the chambers from the extra
heat generated by the lights. Three chambers from each row were used for this study.
Each chamber has four gas ports and cluster valve controllers for gas injection
and sampling. Concentration of CO2, light levels, temperature, and relative humidity can
12
be sampled and measured overtime. The CO2 was supplied as compressed gas, stored in
tanks with valves and sensors connected to a CO2 analyzer to maintain the target
concentration. Light was provided by lamps from three different sources located at the
top of each chamber (1 metal halide of 400 Watts, 1 high pressure sodium of 1000 Watts,
and 1 high pressure sodium of 400 Watts). The relative humidity in the chambers was
regulated by both the air handler and humidifiers, which were placed in strategic points
inside the building for a more uniform distribution of the humidity. Humidifiers were too
large to be placed inside each chamber.
Experimental design. Due to limitations in our facilities in terms of
randomization, a split-split-plot design was arranged for this experiment; air temperature
and CO2 were the main plot and subplot respectively. Hereafter, air temperature will be
called simply “temperature”. The six chambers were exposed to one temperature at a time
(recommended by the literature: 21 ºC). After a destructive analysis of the plants grown
at the first temperature, a sub-optimal temperature was tested (3 ºC lower than
recommended). Three Celsius of reduction in temperature was chosen because we
considered it a realistic number that will not represent a dramatic temperature reduction
from a commercial grower perspective. Both temperatures were replicated to have a total
of 4 runs, each run lasted 4-months. Three out of the six chambers used were enriched
with CO2 (~700 µmol mol-1 CO2; “high”) and the other three were kept at ambient levels
(~390 µmol mol-1; “low”). Our target CO2 concentration was 800 µmol mol-1 CO2, such
level was selected based on the literature, since studies have shown that above 1000 µmol
mol-1 there are not further increases in plant growth compared to concentrations between
13
600-1000 µmol mol-1 (Hicklenton, 1988). Inside each chamber, 4 petunia and 4 zinnia
plants were placed (a total of 48 plants per plot). Each group of petunias and zinnias were
randomly placed in each growth chamber with a factorial of 2×2 which includes 2
nutrition levels (low and high) and 2 insect conditions (presence or absence of
whiteflies). Hereafter, the recommended temperature (21 ºC) will be called “optimal” and
the lower temperature (18 ºC) “sub-optimal”.
Data collection and recording. CO2 was recorded by a H2O/CO2 analyzer
(model LI6262, Licor Biosciences, Lincoln, NE); a pump circulated air from the chamber
to the analyzer where CO2 concentrations were measured. The analyzer used a large
character, black lit LCD display that provided direct readout of the measured values as
well an analog readout that could be connected to a computer for a more detailed
monitoring. The temperatures were measured and recorded using a temperature/relative
humidity sensor (model HMP, Campbell Scientific, Logan, UT) and the light intensity
was measured by sensors located at the canopy level. All data were collected every ten
minutes and recorded overtime by a data logger (Micrologger model CR23X, Campbell
Scientific, Logan, UT).
The temperature and CO2 concentrations as expected experienced some
fluctuations throughout the experiment. The target CO2 concentration for the “low CO2”
treatment was an ambient level (385 µmol mol-1); nonetheless, the average recorded was
391 µmol mol-1. The target for the “high CO2” treatment was 800 µmol mol-1; however,
we recorded an average CO2 concentration of 703 µmol mol-1. On the other hand, the
temperature was stable throughout the duration of the experiment. We obtained an
14
average of 18.1 and 20.9 ˚C for the sub-optimal and optimal treatment respectively
(Appendix A: Table 1-8). The difference between day and night temperatures (DIF) was
also calculated; we obtained averages of 2.34 and 3.87 ºC for the sub-optimal and optimal
temperature respectively (Appendix A: Table 1-8).
Plant material. Petunias (Petunia×hybrida Vilm.), cultivar „Dream Neon Rose‟,
and zinnias (Zinnia elegans L.) cultivar, „Oklahoma White‟ are two common floral crops
grown in the United States. Unlike zinnias whose wholesale value is not reported,
petunias accounted for US$ 110,171,000 in value of all sales at wholesale in the form of
flats, hanging baskets and potted plants in the 15 states reported (USDA-NASS, 2007).
Furthermore, literature mentions that petunias have responded to increased concentrations
of CO2 (Ball, 1991).
Plants were germinated in 200-cell trays at the Entomology greenhouses at
OARDC. For the seedling stage, the temperature was kept at 21.0/15.5 ºC day/night and
watered with 80 ml per day per plant, with a fertilization solution of 100 mg·L-1 N of a
20-10-20 of N, P2O5, K2O fertilizer. Moreover, plants were monitored once a week to
avoid pest contamination to ensure the production of clean plants for the chambers.
Petunias were sowed two weeks earlier than zinnias because at the early stages petunias
grow slower than zinnias. Five weeks after sowing the petunias and three weeks after
sowing the zinnias, both crops were transplanted to a 15.2 cm diameter pots (1 liter
volume) using PROMIX-BX soilless media and placed in the chamber to receive their
respective fertilization and insect treatments.
15
Plant growth and development. Plants were transplanted at a similar size and no
destructive analysis was performed until the end of the experiment. Plant height was
measured from the base of the stem to the highest point observed in the plant canopy. Dry
weight was measured by drying the leaves at 50 ˚C during 72 hours. Plant development
was assessed by counting the total number of flowers and measuring the flower longevity
and the time to flowering which are the most common parameters used to determine plant
development (Hartley et al., 1995; Heineke et al., 1999; Kettenring et al., 2009; Khan et
al., 2008; Purohit and Tregunna, 1974b; Reekie et al., 1997). The first 10 fully opened
flowers were tagged to determine the flower longevity and time to flowering. However,
in the case of zinnias, we could not determine flower longevity because the
inflorescences never reached abscission during the period of observation.
Plant nutrition. At transplant, the fertilization treatments imposed to both
petunias and zinnias were 2.38 g (low) and 3.96 g per container (high) of Osmocote plus
(15-9-12 N, P2O5, K2O). This Controlled Release Fertilizer (CRF) was mixed with the
bottom half soil in the pot to make it more available for the roots. Plants were irrigated as
needed. Five grams of leaf samples were ground and sent for tissue analysis to the
USDA-ARS
Toledo Nutrient
Analysis
laboratory at
the Toledo University.
Concentrations of nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium
(Mg) sulfur (S) and carbon (C) were determined. All nutrient content except N was
determined with inductively-coupled-plasma optical-emission spectroscopy ICP-OES
(Model IRIS Intrepid II, Thermo Corp., Waltham, MA), as for nitrogen analysis, a C-H-N
analyzer (Model 2400, Perkin Elmer, Waltham, MA) was used. Nutrient values obtained
16
in the current experiment were compared with the acceptable values for petunias and
zinnias reported in the Plant analysis handbook II (Mills and Jones, 1997) and lower
values were registered in our experiment.
Insect material. Whiteflies biotype B were reared following the protocol
developed by the Insect Ecology in Controlled Environments laboratory, at the
Entomology department in OARDC (Appendix A: Protocol to rear whiteflies). Since
whitefly colonies are kept in either poinsettias or broccoli plants, it was necessary to
obtain two new colonies in petunias and zinnias to allow for an acclimation period.
Acclimation of insects to petunias and zinnias was important for the insects to adapt to
the new plant material, thus reducing stress by the time of the infestation. Once the
insects were infested in the petunias and zinnias, we allowed the whiteflies to lay eggs for
three days and then we removed all the adults, leaving only the eggs in the plants. After
7-10 days, the eggs hatched and the new individuals started to acclimate in our model
plant for at least two generations.
The acclimated whiteflies were then aspirated for the infestation of the
experimental plants already placed in the growth chambers. For the infestation we
aspirated 6 pair of whiteflies by mouth using plastic tubing (latex tubing amber, VWR
Scientific, Radnor, PA), connected to a glass pipette (53/4 pasteur pipettes, Fisher
Scientific, Pittsburgh, PA). Once the whiteflies were collected, the tip of the pipette was
sealed with parafilm (American National Can, Neenah, WI) to avoid any escape. The
pipettes were taken to the laboratory and the whiteflies sex was identified under the
17
microscope to ensure that 6 females were collected. Whiteflies were carefully released
into a clip-cage attached to the leaf (Fig. 1) by blowing through the pipette. The
infestation was established two weeks after transplanting so the leaf size was large
enough to fit the clip-cage. Even though a study conducted on evaluating effects on insect
clip-cages concluded that physiological and biochemical alterations of leaves can be
produced (Crafts-Brandner and Chu, 1999), we are confident that the three days that we
kept the clip cages in the leaves did not alter our results.
Insect’s oviposition. Eighteen whiteflies per plant were infested (3 leaves with 6
adults per clip-cage). The whiteflies were allowed to lay eggs for three days and then the
adults in the clip-cage were shifted to another leaf. The number of whitefly females
present in each clip-cage was counted before each shift so we could collect the data for
the number of eggs per female per day. We kept shifting clip-cages to new leaves until all
the whiteflies were dead and all countings were recorded in a excel spread sheet.
Nymph development. B. tabaci has 6 developmental stages (egg, 4 nymph
stages and adult). The change in nymph stages was followed to determine nymph
development. We took the first three leaves used to determine oviposition and marked 6
nymphs in first instar only (Fig. 2). We monitored the nymphs every three days and data
was recorded in a spread sheet developed by the laboratory of insect ecology in
controlled environments. Development was followed until the last nymph emerged.
Waiting for the insects to emerge resulted in different plant harvesting times between the
sub-optimal (harvested 13 weeks after transplanting) and optimal temperature (harvested
18
12 weeks after transplanting). From the same spread sheet we collected the information to
determine nymph mortality.
The time from egg hatching to the first nymph emerged, to the 50% of nymphs
emerged and to the 100% of nymphs emerged was determined by using degree-days. The
base threshold temperature for whiteflies (10 ˚C) and the averages day-temperature
registered were used to calculate the degree days. One degree day results when the
average daily temperature is one degree higher than the base temperature threshold. Since
insects are dependent on temperature to develop, degree days have become a common
way to calculate insect development (Kowalsick, 2010). We subtracted the threshold
temperature for whiteflies from the average temperature registered each day to have the
degree days accumulated in one day. The sum of all degree days accumulated per day
from the time of eggs hatching to either, the first nymph emerged, the 50% of nymphs
emerged and the 100% of nymphs emerged were calculated.
Fig.1. Clip cage to confine whiteflies is attached to a petunia leaf
19
Fig.2. First instar whiteflies circled in a petunia leaf. The development of the nymph in
each circled was followed until emergence
Statistical Analysis. The Statistical Analysis Software (SAS® Institute, Cary,
N.C.) was used for data analysis. The PROC-GLM procedure was used to perform the
analysis of variance (ANOVA), which makes comparison of the media using the least
significant difference (LSD) method. The LSD is a common way for comparing
treatments; it computes the smallest significant difference between two means and any
difference larger than such smallest difference is declared significant. We used a
predetermined significance of P≤0.05. Although the results presented here are performed
with GLM we also used PROC MIXED to corroborate our findings. We tested for
homogeneity and normality in all cases. Insect data was transformed using the
logarithmic transformation procedure. Results obtained were graphed using the
SigmaPlot 11.0 software. Each individual chamber within each CO2 concentration was
considered a pseudo-replicate and the error term was pooled since no difference among
blocks (chamber) was found when they were analyzed individually.
20
RESULTS
PETUNIAS
Plant growth and development. Significant differences in plant dry weight were
observed only for the triple interaction of temperature, CO2 and fertilization level. We are
illustrating the higher dry weight obtained in plants treated with high fertilization
regardless of temperature and CO2 treatment (Fig. 3). Plant height indicated significant
effects for the interactions CO2×temperature (P≤0.003) (Fig. 4) and CO2×fertilization
(P≤0.016) (Fig. 5). At optimal temperature and high fertilization, we observed that plants
grown at low CO2 concentrations were taller than those at elevated CO2; however, when
treated with low fertilization rates, plants had similar height. Even though the interaction
between temperature and CO2 was significant for height, it did not influence total dry
weight. Development of plants was significantly affected by treatments. The total number
of flowers of plants at the sub-optimal temperature was higher at high fertilization than
those at low levels of fertilization; whereas within the optimal temperature no difference
was found (Fig. 6).
Across all temperature and insect treatments, there was no difference in flowering
time at the low CO2 level (Fig. 7). Plants took the longest time to flower when they were
grown at high CO2 and optimal temperature regardless of the insect treatment.
Nonetheless, the shortest time to flower was also found on those plants grown at high
CO2, but when plants were exposed to sub-optimal temperature and insects were present
(Fig. 7). While at the sub-optimal temperature the plants fertilized with the low rate
21
flowered earlier than plants with the high rate (P≤0.0255), at the optimal temperature the
fertilization treatment did not have any effect on flowering time (Fig. 8).
Significant effects on flower longevity were observed only for the individual
effects of temperature and CO2. Flowers at the sub-optimal temperature lasted 4.3 days
longer than at optimal temperatures (Fig. 9). At elevated CO2, we also observed a
statistically significant reduction in the flower longevity; however, such reduction was
only by 1 day (Fig. 10).
35
Petunias
a
30
a
Dry weight (g)
ab
a
25
bc
c
20
c
c
15
10
Fertilization level = Low
CO2 level =
High
Low CO2
Low
High CO2
Sub-optimal temperature
High
Low
High
Low CO2
Low
High
High CO2
Optimal temperature
Fig.3. Effects of temperature, CO2 and fertilization levels on petunias‟ dry weight. Plants
were grown in growth chambers at either sub-optimal (18 ˚C) or optimal temperature (21
˚C); low (390 µmol·mol-1) or high (700 µmol·mol-1) CO2 concentrations and low (2.38 g)
or high (3.96 g per plant) fertility levels. Error bars represent the Standard Error of the
mean. Values with the same letter are not significantly different (P≤0.05).
22
24
Petunias
a
b
Height (cm)
22
bc
20
c
18
16
CO2 level =
Low CO2
High CO2
Low CO2
High CO2
Optimal temperature
Sub-optimal temperature
Fig.4. Effects of temperature and CO2 concentration on petunias‟ height. Plants were
grown in growth chambers at either sub-optimal (18 ˚C) or optimal temperature (21 ˚C),
and low (390 µmol·mol-1) or high (700 µmol·mol-1) CO2 concentrations. Error bars
represent the Standard Error of the mean. Values with the same letter are not significantly
different (P≤0.05).
24
Petunias
a
Height (cm)
22
b
b
20
b
18
16
Fertilization level =
CO2 level =
Low
High
Low CO2
High
Low
High CO2
Fig.5. Effects of CO2 and fertilization level on petunia‟s height. Plants were grown in
growth chambers at either low (390 µmol·mol-1) or high (700 µmol·mol-1) CO2
concentrations with a low (2.38 g) or a high (3.96 g per plant) fertilization level. Error
bars represent the Standard Error of the mean. Values with the same letter are not
significantly different (P≤0.05).
23
130
Petunias
a
Total number of flowers
120
110
b
100
b
90
b
80
70
60
Fertilization level =
Low
High
Low
High
Optimal temperature
Sub-optimal temperature
Fig. 6. Effects of temperature and fertilization level on the total number of petunia
flowers. Plants were grown in growth chambers at either sub-optimal (18 ˚C) or optimal
temperature (21 ˚C) and fertilized with either 2.38 g or 3.96 g of fertilizer per plant for
the low and high fertilization levels respectively. Error bars represent the Standard Error
of the mean. Values with the same letter are not significantly different (P≤0.05).
45
Petunias
Time to first open flower (days)
40
a
ab
b
b
35
b
30
b
b
c
25
20
Insect condition =
CO2 level =
Ins+
Ins-
Low CO2
Ins+
High CO2
Sub-optimal temperature
Ins-
Ins+
Ins-
Low CO2
Ins+
Ins-
High CO2
Optimal temperature
Fig. 7. Effects of temperature, CO2 and insect conditions on the time to first open petunia
flower. Plants were grown in growth chambers at either sub-optimal (18 ˚C) or optimal
temperature (21 ˚C), low (390 µmol·mol-1) or high (700 µmol·mol-1) CO2 concentrations
and with or without the presence of whiteflies. Error bars represent the Standard Error of
the mean. Values with the same letter are not significantly different (P≤0.05).
24
38
Petunias
Time to first open flower (days)
36
a
a
34
32
a
30
28
b
26
24
22
20
Fertilization level =
Low
High
Low
Sub-optimal temperature
High
Optimal temperature
Fig. 8. Effects of temperature and fertilization levels on the time to first open petunia
flower. Plants were grown in growth chambers at either sub-optimal (18 ˚C) or optimal
temperature (21 ˚C) and fertilized with 2.38 g (low) or 3.96 g (high) of fertilizer per
plant. Error bars represent the Standard Error of the mean. Values with the same letter are
not significantly different (P≤0.05).
Petunias
a
Average longevity of flowers (days)
14
12
b
10
8
Sub-optimal
Optimal
Temperature level
Fig.9. Effects of temperature on the average longevity of petunia flowers. Plants were
grown in growth chambers at either sub-optimal (18 ˚C) or optimal temperature (21 ˚C).
Error bars represent the Standard Error of the mean. Values with the same letter are not
significantly different (P≤0.05).
25
Petunias
Average longevity of flowers (days)
14
a
b
12
10
8
Low
High
CO2 concentration
Fig. 10. Effects of CO2 concentration on the average longevity of petunia flowers. Plants
were grown in growth chambers at either low (390 µmol·mol-1) or high (700 µmol·mol-1)
CO2 concentration. Error bars represent the Standard Error of the mean. Values with the
same letter are not significantly different (P≤0.05).
Plant nutrient concentration. Total plant nutrient concentrations and its
respective significance level are summarized in Table 1. Although the concentrations of
N were not affected by any of the CO2 treatments, it was significantly affected by
temperature and fertilization levels. At the sub-optimal temperature, the N concentration
was less than at the optimal temperature and consistently, petunias had higher N
concentrations in plants treated with the high fertilization level than those with the low
level (P≤0.03).
Phosphorus concentrations were similar for all treatments except for those plants
grown at low CO2 and optimal temperatures, which higher concentrations than the rest of
26
the treatments (P≤0.02) were detected. The insect presence increased the P content
(P≤0.0065) compared to those plants with no insects.
Potassium and calcium were found in smaller concentrations when plants were
treated with high fertilization levels (P≤0.0001). The concentrations of S on plants treated
with the high fertilization level were similar for the main effect of temperatures, CO2, and
insect treatments. Plants treated with the low fertilization level and sub-optimal
temperatures exhibited the lowest S concentration. No differences were found for Mg
concentrations at any treatment. No effect of elevated CO2 on the C concentration was
found but at the high fertilization level C was higher than at the low level (P≤0.0005).
Significant differences for the C/N ratio were observed for the main effects of
temperature (P≤0.018), fertilization level (P≤0.0001) and CO2 (P≤0.05). At the suboptimal temperature higher C/N ratio was observed, as well as for the elevated CO2 and
the low fertilization rate. The letters assigned to the C/N ratio on Table 1 are based on the
effects of temperature only, since main effects of CO2, and fertilization were also
observed, it was difficult to separate the main effect and assign letters to the result for
each treatment.
27
Sub-optimal temperature
Optimal temperature
Low CO2
Low-fert
High CO2
High-fert
With
Insect
No
Insect
With
Insect
N
1.48d
1.49d
P
0.26c
0.25d
K
Ca
2.51a
2.82a
2.45a
2.36a
Mg
0.44a
0.38a
S
0.82c
0.67c
C
38.73
b
39.56
b
26.95a
Low-fert
Low CO2
High-fert
Low-fert
High CO2
High-fert
No
Insect
With
Insect
1.71c
No
With
Insect
Insect
(% of dry weight)
1.56c
2.50b
2.58b
0.24c
0.26d
0.32a
0.32b
1.45b
2.48b
1.41b
2.52b
2.43a
2.79a
2.76a
2.56a
Low-fert
High-fert
No
Insect
With
Insect
No
Insect
With
Insect
No
Insect
With
Insect
No
Insect
With
Insect
No
Insect
1.84c
1.76c
1.54d
1.39d
0.30c
0.27d
0.28c
0.23d
3.04a
2.95a
2.15b
1.99b
2.74a
2.47a
0.36a
0.29b
0.28c
0.23d
0.28c
0.21d
2.24b
2.51b
1.91b
2.57b
2.50a
2.62a
1.87a
2.40a
1.85b
2.33b
1.91b
2.11b
2.32a
2.45a
2.37a
3.03a
1.83b
1.94b
1.65b
2.50b
0.45a
0.47a
0.40a
0.37a
0.38a
0.43a
0.45a
0.46a
0.49a
0.46a
0.42a
0.48a
0.43a
0.46a
0.85ab
0.82ab
0.66c
0.64c
0.71ab
0.71ab
0.93ab
0.80ab
0.77bc
0.69bc
0.73ab
0.72ab
0.70bc
0.75bc
39.39a
39.46a
39.40b
39.23b
39.87a
39.61a
38.10b
38.68b
39.25a
39.14a
39.52b
37.87b
39.79a
39.23a
22.14a
23.69a
27.56a
28.33a
24.17a
26.08a
16.04b
14.49b
14.77b
18.98b
20.15b
15.55b
16.33b
(ratio)
C/N
26.87a
16.35b
28
Opt=optimal
Fert=fertilization
Table 1. Concentration of macronutrients, carbon and C/N ratios in petunia leaves. Plants were grown in growth chambers at
either sub-optimal (18 ˚C) or optimal temperature (21 ˚C); low (390 µmol·mol-1) or high (700 µmol·mol-1) CO2
concentrations; low (2.38 g) or high (3.96 g per plant) fertility levels and either presence or absence of whiteflies. Numbers
with the same letters in a row are not significant (P≤0.05). No comparison was made between nutrients.
Insect performance. Although petunias were infested with insects, they did not
survive in the plants for long, thus no data was available to evaluate insects performance.
Nevertheless, as showed in Fig. 7, insects affected the flowering time of the plant. The
presence of whiteflies in plants grown at high CO2 and sub-optimal temperature resulted
in the shortest time to flower. All other variables measured produced similar times to
flower, except for those plants at the optimal temperature and elevated CO2, which took
the longest time to flower (Fig. 7).
ZINNIAS
Plant growth and development. We observed a significant difference in dry
weight at the interaction between the fertilization level and insects presence. Plants
grown at high fertilization level experienced an increase of 31% in dry weight over those
grown at low level (Fig. 11), but when plants were infested with insects we found a
decrease of 7.5% in the dry weight (Fig. 12). Although dry weight was not affected by
either temperature or CO2, height of plants grown at the sub-optimal temperature and low
CO2 concentrations was increased compared to those grown at elevated CO2.
Nevertheless, similar height was measured in all plants grown at the optimal temperature
except for those treated with the low CO2 and high fertilization level, which were the
tallest plants (Fig. 13).
The flower longevity and the time to flower were not affected by any treatment;
however the total number of flowers increased by 26% when the fertilization was
increased from low to high level (Fig. 14).
29
35
Zinnias
30
a
Dry weight (g)
25
b
20
15
10
5
0
High
Low
Fertilization level
Fig. 11. Effects of fertilization levels on zinnias‟ dry weight. Plants were grown in
growth chambers at either high (3.96 g) or low (2.38 g per plant) fertility levels. Error
bars represent the Standard Error of the mean. Values with the same letter are not
significantly different (P≤0.05).
30
Zinnias
Dry weight (g)
28
a
26
b
24
22
20
Ins-
Ins+
Insect condition
Fig.12. Effect of the presence or absence of insects on zinnias‟ dry weight. Plants were
grown in growth chambers with or without whiteflies. Eighteen adult female whiteflies
were infested per plant. Error bars represent the Standard Error of the mean. Values with
the same letter are not significantly different (P≤0.05).
30
55
a
Zinnias
Height (cm)
50
45
b
bc
bc
40
bc
bc
c
c
35
30
Fertilization level =
CO2 level =
High
Low
Low
High
Low CO2
High CO2
High
Low
Low CO2
Sub-optimal temperature
Low
High
High CO2
Optimal temperature
Fig. 13. Effects of temperature, CO2 and fertilization levels on zinnias‟ height. Plants
were grown in growth chambers at either sub-optimal (18 ˚C) or optimal temperature (21
˚C), low (390 µmol·mol-1) or high (700 µmol·mol-1) CO2 concentrations and high (3.96
g) or low (2.38 g per plant) fertility levels. Error bars represent the Standard Error of the
mean. Values with the same letter are not significantly different (P≤0.05).
16
a
Zinnias
14
b
Total number of flowers
12
10
8
6
4
2
0
High
Low
Fertilization level
Fig. 14. Effects of fertilization levels on the total number of zinnia flowers. Plants were
grown in growth chambers at either high (3.96 g) or low (2.38 g per plant) fertility levels.
Error bars represent the Standard Error of the mean. Values with the same letter are not
significantly different (P≤0.05).
31
Plant nutrient concentration. The nutrient concentrations are summarized in
Table 2. Fertilization rates affected the concentration of N, P, K, S and the C/N ratios. As
expected, at the high fertilization, concentrations of nutrients increased but only for N, P
and S compared to the low fertilization. Congruently with results in petunias, K was
found in smaller concentration in plants fertilized with the high doses; furthermore, the
C/N ratio also decreased (P≤0.0001) when plants were fertilized with the high level (Fig.
15).
Also in agreement with results found in petunias, the concentration of P was
increased (P≤0.0005) when whiteflies were present in the plant. We also observed a
decrease on the concentration of K (P≤0.0005) and C (P≤0.015) with the presence of
whiteflies (Table 2). The concentration of Ca was affected by the interaction of
temperature and insect treatments (P≤0.04). When insects were present the concentration
of Ca was reduced in those plants at the optimal temperature but not in those at the suboptimal temperature. The lowest Mg concentration was found in plants at the low CO2
concentrations with low doses of fertilizer (Table 2).
32
36
a
Zinnias
34
32
C:N ratio
30
b
b
28
b
26
24
b
b
22
bc
c
20
18
Fertilization level =
CO2 level =
High
Low
Low CO2
High
Low
High CO2
Sub-optimal temperature
High
Low
Low CO2
High
Low
High CO2
Optimal temperature
Fig. 15. Effects of temperature, CO2 and fertilization levels on the carbon-nitrogen ratio
of zinnias. Plants were grown in growth chambers at either sub-optimal (18 ˚C) or
optimal temperature (21 ˚C), low (390 µmol·mol-1) or high (700 µmol·mol-1) CO2
concentrations and high (3.96 g) or low (2.38 g per plant) fertility levels. Error bars
represent the Standard Error of the mean. Values with the same letter are not significantly
different (P≤0.05).
33
Sub-optimal temperature
Optimal temperature
Low CO2
High-fert
High CO2
Low-fert
No
Insect
With
Insect
No
Insect
With
Insect
N
2.07a
1.96a
1.54b
P
0.29b
0.36a
0.24d
K
Ca
1.53b
2.57a
1.99b
2.77a
Mg
1.11a
S
0.35a
C
39.06a
High-fert
Low CO2
Low-fert
High-fert
High CO2
Low-fert
No
Insect
With
Insect
No
Insect
With
No
Insect
Insect
(% of dry weight)
With
Insect
No
Insect
With
Insect
1.69b
1.73a
1.98a
1.48b
1.62b
1.99a
2.09a
1.76b
0.27c
0.27b
0.32a
0.22d
0.29c
0.23b
0.23a
0.21d
2.16a
2.06a
2.00a
2.79a
1.34b
2.46a
2.07ab
2.86a
1.27ab
2.91a
2.23a
2.69a
1.23b
2.77a
1.55b
2.56b
1.40a
0.92b
1.05b
1.19a
1.39a
1.41a
1.20a
1.50a
0.27a
0.25b
0.26b
0.29a
0.34a
0.34ab
0.26b
0.26ab
37.78
b
39.35a
37.89b
37.44a
37.41b
37.19a
36.26b
37.55a
19.52b
27.63a
23.72a
22.21b
19.55b
25.34a
24.04a
High-fert
Low-fert
No
Insect
With
Insect
No
Insect
With
Insect
1.75b
1.90a
1.88a
1.30b
1.26b
0.20c
0.20b
0.21a
0.17d
0.20c
1.73a
2.38a
2.10a
1.96b
1.03b
2.65a
1.43b
2.04b
1.20ab
2.20a
1.40ab
2.53ab
1.52a
1.20b
1.13b
1.32a
1.20a
1.29b
1.36b
0.24ab
0.20b
0.17b
0.24ab
0.21ab
0.14b
0.15b
37.41b
37.68a
37.43b
38.34a
37.67b
37.63a
37.01b
21.31b
26.25a
25.24a
20.47b
20.63b
32.12a
31.44a
(ratio)
34
C/N
19.6ab
20.59b
Fert=fertilization
Opt=optimal
Table 2. Concentration of macronutrients, carbon, and C/N ratios in zinnia leaves. Plants were grown in growth chambers at
either sub-optimal (18 ˚C) or optimal temperature (21 ˚C); low (390 µmol·mol-1) or high (700 µmol·mol-1) CO2
concentrations; low (2.38 g) or high (3.96 g per plant) fertility levels and either presence or absence of whiteflies. Numbers
with the same letters in a row are not significant (P≤0.05). No comparison was made between nutrients.
Insect’s performance. Reducing the optimal temperature by 3 ˚C resulted in a
reduction by 50% in the number of eggs laid per female (P≤0.008) (Fig. 16). Even though
CO2 produced no significant difference on oviposition rate, we consider important to
mention that at the optimal temperature we observed a tendency of an increase in nymph
mortality at elevated CO2; however, at sub-optimal temperatures that tendency was the
opposite (Fig. 17). The only changes observed on the time from egg hatching to nymph
emergence were produced by temperature. We found that when plants were grown at the
sub-optimal temperature, the degree days to emergence of the first nymph, emergence to
the 50% of nymphs
and emergence to the 100% of nymphs (Fig. 18, 19 and 20
respectively) was significantly increased compared to plants grown at the optimal
temperature.
3.0
Zinnias
a
2.5
Eggs/female/day
2.0
1.5
b
1.0
0.5
0.0
Sub-optimal
Optimal
Temperature level
Fig. 16. Effects of temperature on whiteflies oviposition rate (eggs per female per day) of
whiteflies. Plants were grown in growth chambers at either sub-optimal (18 ˚C) or
optimal temperature (21 ˚C). Whitefly‟s eggs were counted every three days during the
adult female‟s lifespan. Error bars represent the Standard Error of the mean. Values with
the same letter are not significantly different (P≤0.05).
35
1.0
Zinnias
a
a
Nymph mortality (percentage)
0.8
b
0.6
b
0.4
0.2
0.0
CO2 level =
Low CO2
High CO2
Low CO2
Sub-optimal temperature
High CO2
Optimal temperature
Fig.17. Effects of temperature and CO2 levels on nymph mortality of whiteflies infested
on zinnias. Plants were grown in growth chambers at either sub-optimal (18 ˚C) or
optimal temperature (21 ˚C) with low (390 µmol·mol-1) or high (700 µmol·mol-1) CO2
concentrations. Whitefly‟s nymphs were circled and mortality was recorded throughout
their developmental time. Error bars represent the Standard Error of the mean. Values
with the same letter are not significantly different (P≤0.05).
300
Degree days to the first nymph emerged
Zinnias
250
a
b
200
150
100
50
0
Sub-optimal
Optimal
Temperature level
Fig. 18. Effects of temperature on the degree days to the first whitefly emerged on
zinnias. Plants were grown in growth chambers at either sub-optimal (18 ˚C) or optimal
temperature (21 ˚C). Whitefly‟s nymphs were circled and their development followed
until emergence. Error bars represent the Standard Error of the mean. Values with the
same letter are not significantly different (P≤0.05).
36
300
Zinnias
Degree days to 50% nymph emergence
a
250
b
200
150
100
50
0
Sub-optimal
Optimal
Temperature level
Fig.19. Effects of temperature on the degree days to the 50% of whitefly emergence on
zinnias. Plants were grown in growth chambers at either sub-optimal (18 ˚C) or optimal
temperature (21 ˚C). Whitefly‟s nymphs were circled and their development followed
until 50% emergence. Error bars represent the Standard Error of the mean. Values with
the same letter are not significantly different (P≤0.05).
350
Degree days to 100% nymph emergence
Zinnias
300
a
b
250
200
150
100
50
0
Sub-optimal
Optimal
Temperature level
Fig. 20. Effects of temperature on the degree days to 100% of whitefly emergence on
zinnias. Plants were grown in growth chambers at either sub-optimal (18 ˚C) or optimal
temperature (21 ˚C). Whitefly‟s nymphs were circled and their development followed
until all nymphs emerged. Error bars represent the Standard Error of the mean. Values
with the same letter are not significantly different (P≤0.05).
37
DISCUSSION
In both petunias and zinnias, the response of dry weight to the CO2 concentration
and temperature treatments was not significant which agrees with the study of (Idso et al.,
1988) who found that, in general, the effects of elevated CO2 on the total biomass weight
were very small. Nevertheless, Reekie et al. (1997) reported an increase in the dry weight
of petunias grown at elevated CO2 which is also congruent with the increase found for
shoot and root biomass at high CO2 levels (Tocquin et al., 2006). Furthermore, a study on
Arabidopsis thaliana also showed an increase in the dry weight at elevated CO2 (Gibeaut
et al., 2001). The fact that we grew our plants in 15.2 cm pots might have affected the
potential effect of CO2 by physically limiting the root expansion. The use of bigger pot
sizes for an accentuated effect of CO2 on physiological aspects of the plant has been
already suggested (Taub and Wang, 2008; Yin, 2002). Furthermore, it is possible that our
plants might have experienced a sink limitation to use the elevated CO2 levels provided.
In a recent study evaluating the photosynthetic responses due to CO2 enrichment
Kirschbaum (2011) mentioned that extra growth can be attained only if the plant has a
way to utilize it, if there is sink limited capacity, increase in photosynthesis will be
difficult to obtain.
The increased dry weight at high fertilization levels obtained for both crops in this
study was an expected outcome and it has been extensively documented as well (Bazzaz,
1990; Frantz and Ling, 2011; Franzaring et al., 2008; Goverde et al., 2002; Schutz et al.,
2008).The shorter plants observed for petunias was similar to the results obtained for
zinnias and also to the results obtained for Lolium perenne cv. „Tove‟ and Phleum
38
pratense cv. „Forus‟ (Saeb and Mortensen, 1995). However, for zinnias the observed
results were in plants grown at high fertilization rates only.
Even though lateral branching was not measured in this experiment, we speculate
based on visual assessment and some literature available (Li et al., 2007; Reekie et al.,
1997) that petunias at the elevated CO2 had more branches, compensating for the short
size resulting in similar dry weights than the taller plants. For instance, Reekie et al.
(1997) demonstrated that the number of branches in petunias was increased when plants
were grown at high CO2. Moreover, Li et al. (2007) reported that lateral grow in tomato
seedlings was enhanced at high CO2 levels; nevertheless such growth was only obtained
when plants were subjected to high nutrient solutions. While our findings in terms of
height were consistent between the two crops, opposite results were reported by Schadler
et al. (2007), who demonstrated that plants (12 species from 10 different taxonomic
families) were taller when grown with elevated CO2 levels.
The increase in the total number of flowers observed in petunias grown at high
fertilization with sub-optimal temperature was consistent with results reported for
Petunia axillaris by Warner (2010) who reported an increased number of flower buds in
plants grown at 14 ºC than in those at 26 ºC . Moreover, they also reported an increased
number of flower buds for P. exerta, and P. integrifolia. In the same study, they
mentioned that nine cultivars of Petunia×hybrida had higher number of flowers and
flower buds at 14 ºC than at 26 ºC. Furthermore, Selander and Welander (1984) also
demonstrated that Primula vulgaris cv. „Ducat‟ produced fewer flowers as temperature
increased from 12 to 15 ºC. On the other hand, the increased number of flowers observed
in zinnias grown at the high fertilization rate was expected because the availability of
39
nutrients to allocate biomass in flower tissues can lead to an increase in flower number.
However, in a recent study conducted with petunias, it was reported that less biomass was
allocated as fertility was increased (Frantz and Ling, 2011).
The increase observed in the flower longevity of petunias when temperature was
reduced from optimal to sub-optimal is similar to that for Easter Cactus (Rhipsalidopsis
gaertneri) var „Red Pride‟ where the longevity of flowers at 18 ºC was 3 days greater
than plants exposed at 24 ºC (Hartley et al., 1995). The report of Reid (1991) that the
respiration rate is reduced when temperature is decreased resulting in reduced plant
metabolism may help explain the increase found in flower longevity.
While no significant differences were observed across treatments on the time to
flower in zinnias, the reduction in time to flower reported for petunias at high CO2 and
sub-optimal temperature agreed with results found by Reekie et al. (1997) who observed
that plants flowered 5.1 days earlier at elevated than at ambient CO2. In this case the
elevated CO2 overcame the potential detrimental effects of low temperature. It is
expected that flower buds can be delayed or inhibited because of the cooling effect of
temperature (Reid, 1991).
It has been suggested that interaction between CO2 and
phytocromes might explain the effects of elevated CO2 on developmental time such as
flowering time (Purohit and Tregunna, 1974a); hence, helping explain the positive
changes in flower development found at elevated CO2 even when temperature was
reduced.
In contrast with the increase in the time to flower found for Liatris ohlingerae by
(Kettenring et al., 2009), when insects were present, we observed a reduction in such
40
time. We suggest that such reduction in flowering time was due to a response of the plant
to develop faster because of stress caused by insect presence. However, not many reports
are available of how plants perceive insects attack, not even in terms of defensive
mechanisms which is the more studied area in herbivore-plant interactions (Wu and
Baldwin, 2010). The effect of fertilization on the time to flower was evident at the lower
temperature only, where plants took longer to flower when they were fertilized with the
high rate. One possible explanation is that lower temperature decreased enough the
photosynthetic rate that resulted in the increased time to flower (Reid, 1991). Another
explanation might be that high nutrition produced a comfort zone for the plant resulting
in delaying the time to reproduce thus directing resources to growth instead. It has been
demonstrated that flowering in onion (Alliun cepa L.) was delayed in plants fertilized
with the high N treatment (Diaz-Perez et al., 2003).
Another point to be addressed is the effect that the difference between day and
night temperatures (DIF) (Hwang et al., 2005; Kaufmann et al., 2000; Moe et al., 1995)
may have on the plant growth and development. We have calculated the DIF for the main
plots (temperature) and we recorded positive DIF of 2.3 and 3.9 ˚C for the sub-optimal
and optimal temperatures, respectively. Even though both DIF are positive, it is not
possible to know if the 1.6 ˚C difference between them significantly affected plant height.
We suggest that more work in this area is needed.
The nutrient concentration was variable across all the treatments measured for
both crops investigated. Even though our values were below the minimum acceptable
values (Mills and Jones, 1997), suggestions have been made that such values can be
different depending of the environmental condition that plants are exposed (Taub and
41
Wang, 2008). Moreover, visually we did not observe any nutritional deficiency
symptoms in the plants. While most studies have found a decrease in the N content in
leaves of plants grown at high CO2 (Cotrufo et al., 1998; Curtis and Wang, 1998;
Goverde et al., 2002; Korner and Challa, 2003; Taub and Wang, 2008; Yin, 2002), in this
experiment, petunias and zinnias both had N concentrations not significantly affected by
elevated CO2. Other studies have also reported similar N concentration across CO2
treatments. For instance, Liu et al. (2010) in an analysis of shoot and roots in water
hyacinth (Eichhornia crassipes) no significant differences were found in the total N when
plants were exposed to either ambient or elevated CO2. Furthermore, no significant
difference was observed in the N content for sainfoin plants (Onobrychis viciaefolia)
when grown at elevated CO2 (Zhou and Shangguan, 2009).
The reduction of N in petunias‟ leaves observed in our experiment at the suboptimal temperature may be the consequence of reduced photosynthetic rates (Sage et al.,
1995); thereby, reducing the uptake of this nutrient. In contrast, Ambebe et al. (2009)
investigated the interactive effects of soil and temperature on nutrient availability and
found that seedlings of white birch (Betula papyrifera Marsh) at lower temperature had
similar N concentrations in the leaves than the rest of the temperatures measured in the
study. N concentrations were higher at the high fertilization levels for both petunias and
zinnias, which suggest a positive relation between the fertilization level and the
concentrations of N in the leaves for his two crops.
The higher P concentration at low CO2 and optimal temperatures found in
petunias is attributed more to the effect of the temperature rather than CO2. It is known
that plants at higher temperatures, although lower than 35 ˚C, will result in higher
42
photosynthetic rates (Long, 1991; Sage et al., 1995), which may result in a higher P
uptake (Lukac et al., 2010). The few studies found of the effect of CO2 on P
concentration reported similar concentrations for sweetgum (Liquidambar spp; Johnson
et al., 2004) and poplar trees (Populus spp; Khan et al., 2008) between elevated and
ambient CO2. In both species, P was increased because of whiteflies‟ presence. Although
no similar results were found to compare with our study, a decrease in P due to insect
pressure has been previously documented. For example, Yin et al. (2005) investigated
changes in the P and found that feeding of the rice leaffolder (Cnaphalocrocis medinalis)
caused a decrease in the uptake of this nutrient. The number of nymphs infested in our
study may not be a strong argument to conclude that whiteflies feeding caused a
reduction in P concentration as demonstrated by Yin et al. (2005); however, no literature
was found regarding the effects of insect presence on P in terms of chemical signals as
the plant response. Thus, we suggest investigating the signal responses within the plant
tissues that can lead to either an increase or decrease in nutrient concentrations.
The reduced concentrations in K and Ca found when plants were treated with high
fertilization is similar to results presented in a study conducted with cabbages and
broccoli; K and Ca decreased if N supply was increased (Sorensen, 1999). Furthermore,
the concentration of K in the stems of tomato seedlings was also lower in plants grown at
higher nutrient solutions (Li et al., 2007). The reduction of K in both petunias and zinnias
and the reduction of Ca in petunias treated with high fertilization levels possibly can be
explained by a revision of the dilution hypothesis which states that reduction of N is due
to an accumulation of non-structural carbohydrates (Taub and Wang, 2008).
43
The decrease that insects produced on the Ca concentration for zinnias has also
been demonstrated in the meta-analysis study with trees (Nykanen and Koricheva, 2004);
however, in our study the effect was only observed with optimal temperature. Since no
literature was found regarding the interaction of temperature and insects affecting Ca
concentration we speculate that at the optimal temperature the insect will experience a
faster metabolic rate resulting in more consumption of this nutrient, thereby resulting in
smaller concentration in the plant tissue. Although (Nykanen and Koricheva, 2004)
documented that the decrease in Ca, which has low mobility, may be due to the loss of
tissue. Nonetheless, whiteflies which are sap-sucking insects can extract liquids from the
plant, enough to cause significant nutritional damage (Wu and Baldwin, 2010). While in
petunias Mg did not manifested any significant difference across treatments, in zinnias,
the lowest Mg concentration was observed in plants exposed to low CO2 and low
fertilization which also agrees with results presented by (Nykanen and Koricheva, 2004).
Consistent with results observed by Li et al. (2007), no significant differences
were found in C concentrations in both crops when exposed to elevated CO2. A study
carried out with sainfoin (Onobrychis viviaefolia) indicated that C can present variable
responses to elevated CO2, even within the same plant; the study revealed that at high
CO2 the C in stems and roots decreased by 24.2% and 7.2% respectively, whereas for
leaves no significant difference were reported (Zhou and Shangguan, 2009). Carbon, like
P concentration, also decreased when whiteflies were present but only for zinnias. The
decrease in S concentration at the lower temperatures in petunias indicates that S
accumulation in the leaf tissue is slower if temperature is reduced; in zinnias though, the
effect on S was given by the fertilization treatment only. The increase observed in the
44
C/N ratio in our study cannot be attributed to a decrease in the N concentration or an
increase in the C content since no difference was found for the main effect of CO2 on
either C or N content. An increase in the C/N ratio was also observed in the study
conducted in plant herbivore interaction at elevated CO2 (Himanen et al., 2008; Zhou and
Shangguan, 2009), which contrast with the unchanged C/N ratio found by Johnson et al.
2004). Even though we neither, measured the whiteflies consumption nor observed
significant depletion of N concentration, it has been documented that an increase in C/N
can result from a depletion of N due to the consumption of leaves by insects (Cotrufo et
al., 1998).
Although petunias were infested with insects, the insects did not survive in the
plants, thus no data was collected to evaluate insects performance. We speculate that the
one of the reasons for the early mortality of the adults might be the presence of steroidal
compounds present in some petunia cultivars, such compounds are called petuniasterones
and it has been demonstrated to be harmful to insects (Elliger and Waiss, 1989). For
example, in a study on tomato plants many larvae of the lepidopteran (Heliothis zea
Boddie) die when they were exposed to the crude extracts of these compounds (Elliger
and Waiss, 1989). We also speculate that in addition to the insecticide compounds that
petunias produce, some management techniques might have caused negative effects on
insect ovipostion and development; the manipulation during the infestation process and
being enclosed in a pipette could have stressed the whiteflies, thereby affecting their
performance. Moreover, one of the first responses of petunias to water stress is the
production of a sticky compound in the leaves, which may also have affected the
whiteflies survival.
45
In zinnias, on the other hand, the decrease in whiteflies‟ oviposition observed by
the reduction in temperature was an expected outcome and has been previously
documented (Wang and Tsai, 1996). The effect of CO2 on insect oviposition and nymph
development in zinnias was not significant. Nevertheless, studies at elevated CO2 have
shown a decrease in the growth and development of herbivores (Bezemer and Jones,
1998). The effect of elevated CO2 on nymph mortality was observed only at the optimal
but not at the sub-optimal temperature. Nymph mortality was also increased in a study
evaluating the response of three generations of Neophilaenus lineatus (Hemiptera)
infested on plants exposed to high CO2 concentrations. The researchers concluded that
development of nymphs was delayed by elevated CO2 (Brooks and Whittaker, 1999). The
increase in the degree days observed at the sub-optimal temperature was also expected
and indicates that development of whiteflies took longer to emerge at the sub-optimal
than at the optimal temperature. Our results on nymph development were consistent with
the study conducted with the parasitoid (Amitus benneti) of the silver leaf whitefly; it was
reported that developmental time decreased from 72 days to 42 days when temperature
was increased from 15 to 20 ºC (Drost et al., 1999).
46
CONCLUSION
Petunias
The effects of temperature, CO2 and fertilization levels on plant growth and
development were variable in this chamber study. Although plants were shorter at
elevated CO2, it did not affect the plant‟s dry weight. Similar results obtained for dry
weight and plant height at both temperatures allow us to conclude that a reduction in the
growing temperature in petunias can be done without affecting growth. Moreover, if a
high fertilization program is implemented, a further increase in the dry weight can be
obtained. An increased number of flowers, longer flower longevity and shorter time to
flower were observed by enriching with CO2 and reducing the growing temperature.
In general, elevated CO2 did not have any effect on plant nutrient concentrations.
While at the high fertilization level the concentrations of N and C were higher, the
concentrations of K and Ca were lower compared to the low fertilization level.
Concentrations of P, Mg and S remained unchanged across all treatments, except that P
responded to the insect presence when plants were exposed to the interaction of CO2 and
temperature. To evaluate insect oviposition and nymph development is recommended to
test more susceptible cultivars to avoid early mortality of adults.
Zinnias
The increase of 31% in dry weight in zinnias was given mainly by the increase in
the fertilization level. Even though at the elevated CO2 we found the shortest plants, it did
not have any effect on the total dry weight; furthermore, plant development was not
47
affected unless treated with high fertility, in which case an increased number of flowers
were observed. The concentration of nutrients in zinnias varied depending on the
treatment. In general the nutrient concentrations responded mainly to the fertilization
level by either an increase or a decrease of the concentration in the leaf. Though,
responses to insect pressure or temperature changes were observed for P, Ca, Mg and C.
As it was expected, the sub-optimal temperature decreased oviposition rate in
whiteflies. It also increased the degree days to the first nymph emerged, to the 50%
emerged and to the 100% emerged. Moreover, no effects of CO2 and/or fertilization
treatments were observed on any of the variables measured corresponding to insect
performance.
For both crops, taking into consideration 1) that plants were shorter at elevated
CO2 but dry weight was not negatively affected, 2) that development of petunias at
enriched CO2 /sub-optimal temperature conditions can be similar or faster than that at
ambient CO2/optimal temperatures, 3) that no significant reduction in nutrient
concentration was observed, and 4) that insects‟ oviposition and development were
negatively impacted; the proposed alternative of CO2 enrichment accompanied with a
reduction in temperature may achieve potential energy savings and accomplish reductions
in pesticide applications in the floriculture industry. We have demonstrated with this
study that the predicted expectations of compensating with CO2 enrichment the potential
slowdown in growth and development by a reduced temperature can be achieved.
48
CHAPTER 3
The effect of elevated CO2 on growth, development, nutrient concentrations, and
insect performance of plants grown in a greenhouse at sub-optimal temperature
INTRODUCTION
The greenhouse industry faces economic and environmental challenges that force
growers to develop new sustainable alternatives to produce crops (Omer, 2008). The
reduction in energy consumption to increase profits while meeting the market standards
for plant quality is difficult to achieve, but through an understanding of the proposed
alternative in the current study, substantial improvement towards energy savings can be
accomplished.
One of the first reports on CO2 enrichment in greenhouses was published 1897;
the CO2 released by fresh manure placed inside the greenhouse induced plants to grow
faster than those plants treated with old manure (Bauerle and Kimball, 1984). The
floriculture industry followed the vegetable greenhouse industry in using CO2 enrichment
during the photoperiod as a regular practice to enhance dry matter production at
greenhouse environments (Bauerle and Kimball, 1984).
For a specific plant species, a “typical” growing condition in the greenhouse is
considered when plants are grown at ambient CO2 concentration (385 µmol·mol-1) and
49
the recommended growing temperature. Based on the literature (Ball, 1991), the
recommended “optimal” temperature to grow petunias and zinnias is 21 ˚C. To reduce
energy consumption, a modified growing condition is proposed in this experiment;
increasing air CO2 concentrations (800 µmol·mol-1) while reducing the temperature by 3
˚C from the “optimal” growing temperature.
If successful this technique would be applicable to geographic areas where
greenhouse heating is needed but ventilation is not required. In our growth chamber study
(See chapter 2), conclusions were reached regarding the effect of CO2, temperature,
fertilization rates and insect presence on plant variables such as plant dry weight, plant
height, flowering development, and whiteflies‟ oviposition and development. While
carrying studies in growth chambers provides an excellent environmental control,
greenhouse studies are a more realistic approach closer to what growers are commercially
implementing. To validate results obtained in the growth chamber experiment, the
objective of this greenhouse study was to determine the effect of CO2 enrichment on the
growth, development, plant nutrient concentration and insect performance of petunias and
zinnias grown at sub-optimal temperature.
50
MATERIALS AND METHODS
Plant material, statistical analysis as well as assessment of plant growth and
development, plant nutrition, insect‟s oviposition, and nymph development were identical
to the chambers experiment (please refer to chapter 2).
Facilities. This study was conducted from January to April of 2010 in the
greenhouses located at the department of Food Agricultural and Biological Engineering
at the OARDC, Wooster campus. Two greenhouses compartments were needed to carry
out this experiment. Each compartment covered a total area of 168 m2, of which only 50
m2 were used to conduct our experiment. The side walls of the greenhouses are built with
polycarbonate and the roof with double layered-inflated polyfilm. The insulation of the
greenhouse goes 0.6 m deep into the ground to ensure a more stable temperature into the
compartment especially during spring and autumn when fluctuations can be very
dramatic in the US Midwest region.
One of the compartments was equipped with a natural-gas burner (Johnson Gas
Appliance Company, Cedar Rapids, IA) to supply CO2. The concentration was read with
a CO2 controller model iGS-061 (Nova Biomatique, La Procatiere, Canada). This device
does not record data overtime, thus data read by the CO2 controller was written in a
printed spread sheet. Each day, two or three observations of CO2 values were recorded,
then such data was averaged using excel software. The burner enriched CO2 into the
greenhouse through the combustion of natural gas and it was activated during the
photoperiod only. One of the disadvantages of this method of enrichment is that an
51
incomplete combustion of the natural gas may result in production of ethylene that can
cause early flower senescence. A jet tube of 61cm of diameter (BFG supply Co., Burton,
OH) located at the top of the greenhouse helped distribute the CO2 gas more uniformly
throughout the compartment gradient. Temperature was controlled by a thermostat with
sensors located in the middle section of each greenhouse compartment. However,
HOBOS® data logger devices (Onset Computer Corporation, Southern, MA) were also
placed at canopy level to measure temperature and relative humidity. A CO2 hand-held
meter (Vaisala MI70, Woburn, MA) was used to record the CO2 values in the
compartment set at ambient CO2 concentration.
Experimental design. The objective of this design was to replicate in one of the
compartments “typical” growing conditions while establishing our modified growing
conditions in the other compartment. Two “CO2/temperature combinations” were
established; one of the greenhouses was set at ambient CO2 concentration (385
µmol·mol-1) and “optimal” growing temperature (21˚C) identified here as the
“lowCO2/OT”. Whereas in the other compartment, we increased air CO2 (800 µmol·mol1
) and kept a sub-optimal temperature of (3 °C lower than “optimal”); named here
“highCO2/ST” environmental setting. Hereafter, the recommended temperature to grow
our experimental plants will be referred as “optimal” and the lower temperature as “suboptimal”.
The experimental design used for both greenhouse compartments was a split plot
with 8 blocks in each plot. In total 8 plants (4 petunias and 4 zinnias) were placed per
52
block to constitute a total of 64 plants in each compartment. For each crop, two nutrition
levels (optimal and supra-optimal), and two insect conditions (presence or absence)
comprised our factorial of 2×2 (refer to chapter 2 for details regarding the nutrition
treatments).
Data collection and recording. Readings from the CO2 controller were recorded
manually for the compartment that was enriched with CO2. In the other compartment,
data were recorded every 15 minutes by the CO2 hand-held meter; these data were
downloaded on a weekly basis to monitor the concentration and make adjustments as
needed. Temperature and relative humidity data were measured every 15 minutes and the
averages stored in HOBO® dataloggers. As needed, the thermostat was adjusted to keep
the highCO2/ST compartment 3 ºC cooler that the lowCO2/OT compartment. As
expected, due to external conditions, there were deviations in the actual greenhouse CO2
concentrations and temperature levels that needed periodic corrections. The average CO2
and temperature recorded in the highCO2/ST compartment during the experiment were
786 µmol·mol-1 and 21.9 ºC respectively, whereas in the lowCO2/OT compartment the
averages recorded were 397 µmol·mol-1 and 25.6 ºC. The difference between day and
night temperatures (DIF) was also calculated; we obtained averages of 5.77 and 7.54 ºC
for highCO2/ST and lowCO2/OT respectively (Appendix A: Table 9-10).
53
RESULTS
PETUNIAS
Plant growth and development. No significant effects of CO2, temperature or
insect presence on plant growth were observed. Dry weight and height were only affected
by the fertilization treatment; plants treated with high fertilization had a 25% increase in
dry weight (P≤0.001) and a 13% increase in height (P≤0.0024) compared to plants treated
with low doses of fertilizer (Fig. 21 and 22).
In terms of plant development, an increase of 21% in the total number of flowers
(P≤0.0046) was observed in plants grown at highCO2/ST (Fig. 23). Furthermore, an
increase of 20% in the total number of flowers was observed in plants fertilized with the
high rates (Fig. 24). While an increase in the total number of flowers in plants exposed at
highCO2/ST was observed, flower longevity was reduced when petunias were exposed to
the same CO2/temperature combination (Fig 25). The longevity of flowers at elevated
CO2 and sub-optimal temperature was 2 days shorter than those at low CO2 with optimal
temperatures (P≤0.003), which represents a 27% reduction in flower longevity (Fig 25).
In addition to the reduction in longevity, petunias exposed to elevated CO2 and suboptimal temperatures, took slightly longer to flower than those at lowCO2/OT (P≤0.05)
(Fig. 26). However, such difference in time to flower was only 1.2 days.
54
30
Petunias
a
28
Dry weight (g)
26
24
b
22
20
18
16
High
Low
Fertilization level
Fig.21. Effects of fertilization levels on petunias‟ dry weight. Plants were grown in
greenhouses and fertilized at either a low (2.38 g) or a high (3.96 g per plant) fertility
level. Error bars represent the Standard Error of the mean. Values with the same letter are
not significantly different (P≤0.05).
30
Petunias
a
25
Height (cm)
b
20
15
10
Low
High
Fertilization level
Fig. 22. Effects of fertilization levels on petunias‟ height. Plants were grown in
greenhouses and fertilized at either a low (2.38 g) or a high (3.96 g per plant) fertility
level. Error bars represent the Standard Error of the mean. Values with the same letter are
not significantly different (P≤0.05).
55
150
Petunias
140
Total number of flowers
a
130
120
b
110
100
90
low CO2/OT
high CO2/ST
CO2/temperature combination
Fig.23. Effects of the CO2/temperature combination on the total number of petunia
flowers. Plants were grown in greenhouses at either high CO2 and sub-optimal
temperature (highCO2/ST; 786 µmol·mol-1/21.9 ºC) or low CO2 and optimal temperature
(lowCO2/OT; 397 µmol·mol-1/ 25.6 ºC). Error bars represent the Standard Error of the
mean. Values with the same letter are not significantly different (P≤0.05).
150
Petunias
a
Total number of flowers
140
130
120
110
b
100
90
Low
High
Fertilization level
Fig.24. Effects of fertilization levels on the total number of petunia flowers. Plants were
grown in greenhouses at either low (2.38 g) or high (3.96 g per plant) fertility levels.
Error bars represent the Standard Error of the mean. Values with the same letter are not
significantly different (P≤0.05).
56
10
Petunias
Average longevity of flowers (days)
a
9
8
b
7
6
low CO2/OT
high CO2/ST
CO2/temperature combination
Fig. 25. Effects of the CO2/temperature combination on the average longevity of petunia
flowers. Plants were grown in greenhouses at either high CO2 and sub-optimal
temperature (highCO2/ST; 786 µmol·mol-1/21.9 ºC) or low CO2 and optimal temperature
(lowCO2/OT; 397 µmol·mol-1/ 25.6 ºC). Error bars represent the Standard Error of the
mean. Values with the same letter are not significantly different (P≤0.05).
69
Time to first open flower (days)
Petunias
68
a
67
b
66
65
low CO2/OT
high CO2/ST
CO2/temperature combination
Fig.26. Effects of the CO2/temperature combination on the time to first petunia open
flower. Plants were grown in greenhouses at either high CO2 and sub-optimal
temperature (highCO2/ST; 786 µmol·mol-1/21.9 ºC) or low CO2 and optimal temperature
(lowCO2/OT; 397 µmol·mol-1/ 25.6 ºC). Error bars represent the Standard Error of the
mean. Values with the same letter are not significantly different (P≤0.05).
57
Plant nutrient concentration. Total petunia nutrient concentrations and their
respective significance levels are summarized in Table 3. N concentration was almost 7%
higher (P≤0.0178) in plants grown at highCO2/ST than in plants grown at lowCO2/OT.
Furthermore, N was increased by 23% when fertilization was increased from low to high.
In plants grown at highCO2/ST, the concentration of P, similar to that of N, increased by
7% compared to that of petunias grown at lowCO2/OT. Across all treatments, the
concentrations of K, Ca as well as the C/N ratio remained unchanged.
Results observed for Mg and S were similar at both, highCO2/ST and
lowCO2/OT; however, when fertilization was increased from low to high, an increase in
Mg and S of 15% was observed. The interaction between CO2 concentration, air
temperature and fertilization rate significantly affected C concentration; only at the high
fertilization treatment, concentrations of C in plants exposed to lowCO2/OT were higher
than plants at highCO2/ST (P≤0.01).
58
High CO2/Sub-optimal temperature
Low-fert.
Low CO2/Optimal temperature
High-fert.
With Insect
No Insect
With Insect
N
P
K
Ca
Mg
S
C
1.97b
0.30a
2.38a
2.25a
0.39b
0.65b
38.7ab
1.93b
0.30a
2.21a
2.01a
0.38b
0.59b
39.38ab
2.47a
0.34a
2.32a
2.01a
0.46a
0.69a
38.73b
C/N
19.91a
20.78a
15.97a
Low-fert.
No Insect
With Insect
(% of dry weight)
2.39a
1.92d
0.32a
0.30ab
2.06a
2.57a
2.18a
2.07a
0.48a
0.39b
0.79a
0.58b
38.42b
38.77ab
High-fert.
No Insect
With Insect
No Insect
1.77d
0.28b
2.27a
2.10a
0.40b
0.58b
38.62ab
2.27c
0.29b
2.11a
2.01a
0.46a
0.68a
39.12a
2.18c
0.28b
2.12a
1.75a
0.40a
0.59a
39.55a
21.97a
17.56a
18.56a
(ratio)
16.18a
20.28a
Opt=optimal
Fert=fertilization
Table 3. Concentration of macronutrients, carbon, and C/N ratios in petunia leaves.
Plants were grown in greenhouses at either high CO2 and sub-optimal temperature
(highCO2/ST; 786 µmol·mol-1/21.9 ºC) or low CO2 and optimal temperature
(lowCO2/OT; 397 µmol·mol-1/ 25.6 ºC). Plants were either infested or not infested with
whiteflies and treated with low (2.38 g) or high (3.96 g per plant) fertility levels.
Numbers with the same letters in a row are not significant (P≤0.05). No comparison was
made between nutrients.
Insect performance. Similar to the experiment in the growth chamber, whiteflies
in petunias did not survive long enough to collect data to evaluate insect performance. In
the chamber study, effects of insects on the nutrient concentrations were reported, but in
the greenhouse experiment, no main effects or interactions were observed.
ZINNIAS
Plant growth and development. The dry weight of zinnias was significantly
affected by the interaction between CO2/temperature combination and insect presence
(Fig. 27). While in general the presence of insects reduced the dry weight at both
59
CO2/temperature combinations, the effect was more accentuated at the highCO2/ST than
at the lowCO2/OT setting (P≤0.01). We did not observe any difference of the main effects
of either, CO2/temperature combinations or fertilization rates on plant dry weight.
Similarly with results obtained for dry weight, height was similar across all
environmental and fertilization treatments, but it was different in those plants exposed to
insects (Fig. 28). Zinnias infested with whiteflies were 14% shorter than those plants
where whiteflies were absent.
A triple interaction between CO2/temperature combination, fertilization, and
insect presence was observed (Fig.29). On average, zinnias grown at highCO2/ST
conditions produced fewer flowers than plants at lowCO2/OT conditions. The total
number of flowers was not affected by the main effect of high fertilization. However,
either at highCO2/ST or at lowCO2/OT plants with the high fertilization always had more
flowers than at low fertilization, regardless of the insect treatment. The presence of
insects reduced the number of flowers in zinnias exposed to the highCO2/ST
environmental condition only. Fewer flowers were produced at high than at the low
fertilization level (Fig.29). No effect of CO2, fertilization or insect was observed for the
variables of flower longevity and time to flower.
60
40
Zinnias
38
a
36
Dry weight (g)
ab
34
bc
32
30
c
28
26
Ins-
Ins+
high CO2/ST
Ins+
Ins-
low CO2/OT
Fig. 27. Effects of the CO2/temperature combination and insect condition on zinnia‟s dry
weight. Plants were grown in greenhouses at either high CO2 and sub-optimal
temperature (highCO2/ST; 786 µmol·mol-1/21.9 ºC) or low CO2 and optimal temperature
(lowCO2/OT; 397 µmol·mol-1/ 25.6 ºC). Zinnias were infested with eighteen adult
whitefly females per plant. Error bars represent the Standard Error of the mean. Values
with the same letter are not significantly different (P≤0.05).
76
Zinnias
74
a
72
Height (cm)
70
68
66
64
b
62
60
58
56
Ins-
Ins+
Insect condition
Fig. 28. Effects of the presence or absence of insects on zinnias‟ height. Plants were
grown in greenhouses with or without whiteflies. Eighteen adult whitefly females were
infested per plant. Error bars represent the Standard Error of the mean. Values with the
same letter are not significantly different (P≤0.05).
61
24
Zinnias
a
Total number of flowers
22
ab
20
18
bc
16
bc
bc
cd
cd
14
d
12
10
Fertilization level =
Insect condition =
Low
High
Low
Ins-
High
Ins+
high CO2/ST
Low
High
High
Low
Ins-
Ins+
lowCO2/OT
Fig. 29. Effects of the CO2/temperature combination, insect condition and fertilization
level on the total number of zinnia flowers. Plants were grown in greenhouses at either
high CO2 with sub-optimal temperature (highCO2/ST; 786 µmol·mol-1/21.9 ºC) or low
CO2 with optimal temperature (lowCO2/OT; 397 µmol·mol-1/ 25.6 ºC). Plants were either
infested or not infested with whiteflies and treated with low (2.38 g) or high (3.96 g per
plant) fertility levels. Error bars represent the Standard Error of the mean. Values with the
same letter are not significantly different (P≤0.05).
Plant nutrient concentration. Total leaf nutrient concentrations in zinnias and
their respective significance level are summarized in Table 4. Very few significant
differences were found in the nutrient concentration for zinnias. N increased by 24%
when fertilization was increased from low to high level. No changes were observed for
the concentration of P, Ca and Mg across any treatment. Potassium concentration was
14% higher in zinnias infested with whiteflies than those plants with no whiteflies
(P≤0.0353). Main effects of fertilization and CO2/temperature combination were
observed on S concentrations; an increase of 41% in the concentration of S was observed
in zinnias treated with high fertilization compared with zinnias treated with low
fertilization. Moreover, we found that S was 28% higher in zinnias grown at highCO2/ST
than at low CO2/OT (P≤0.0147). The concentration of C, similar to that of P, Ca and Mg
62
was also similar across all treatments. The triple interaction observed for the C/N ratio
revealed that in general, higher values were obtained in those plants fertilized with low
levels. While a high C/N ratio was expected in plants infested with whiteflies because of
an increase in the nutrient consumption, no clear trends were observed for insect
treatment in the current experiment (Fig. 30).
34
Zinnias
a
32
C:N ratio
30
28
bc
b
bc
bc
26
c
c
24
c
22
20
Fertilization level =
Insect condition =
Low
High
High
Low
Ins-
Ins+
high CO2/ST
High
Low
High
Low
Ins-
Ins+
lowCO2/OT
Fig. 30. Effects of the CO2/temperature combination, insect condition and fertilization
level on the carbon-nitrogen ratio of zinnias. Plants were grown in greenhouses at either
high CO2 with sub-optimal temperature (highCO2/ST; 786 µmol·mol-1/21.9 ºC) or low
CO2 with optimal temperature (lowCO2/OT; 397 µmol·mol-1/ 25.6 ºC). Plants were either
infested or not infested with whiteflies and treated with low (2.38 g) or high (3.96 g per
plant) fertility levels. Plants were either infested or not infested with whiteflies and
treated with low (2.38 g) or high (3.96 g per plant) fertility levels. Error bars represent the
Standard Error of the mean. Values with the same letter are not significantly different
(P≤0.05).
63
High CO2/Suboptimal temperature
Low-fert.
Low CO2/Optimal temperature
High-fert.
Low-fert.
No Insect
With Insect
No Insect
With Insect
No Insect
(% of dry weight)
N
P
K
Ca
Mg
S
C
1.32b
0.19a
1.57b
2.40a
1.41a
0..21b
36.21a
1.40b
0.21a
1.87a
2.32a
1.45a
0.17b
36.60a
1.61a
0.22a
1.35b
2.05a
1.56a
0.24a
37.17a
1.72a
0.21a
1.66a
2.04a
1.51a
0.28a
37.17a
C/N
27.49b
26.55bc
23.19c
21.86c
High-fert.
With Insect
No Insect
With Insect
1.30b
0.18a
1.77b
2.68a
1.77a
0.14d
35.50a
1.19b
0.22a
2.04a
2.04a
1.24a
0.14d
36.88a
1.54a
0.21a
1.68b
1.84a
1.30a
0.17c
37.53a
1.59a
0.22a
1.71a
2.36a
1.48a
0.26c
36.70a
27.49bc
31.43a
25.06bc
23.39c
(ratio)
Opt=optimal
Fert=fertilization.
Table 4. Concentration of macronutrients, carbon, and C/N ratios in zinnia leaves. Plants
were grown in greenhouses at either high CO2 and sub-optimal temperature
(highCO2/ST; 786 µmol·mol-1/21.9 ºC) or low CO2 and optimal temperature
(lowCO2/OT; 397 µmol·mol-1/ 25.6 ºC). Plants were either infested or not infested with
whiteflies and treated with low (2.38 g) or high (3.96 g per plant) fertility levels.
Numbers with the same letters in a row are not significant (P≤0.05). No comparison was
made between nutrients.
Insect performance. When zinnias were grown at highCO2/ST, whitefly‟s
oviposition rate was reduced by 44% compared to those grown at lowCO2/OT (Fig 31).
However, no effect of fertilization treatment was observed. Congruently with results in
oviposition, increases in nymph mortality were also observed; at highCO2/ST, mortality
was 67% whereas at lowCO2/OT mortality was only 49% (Fig 32). While the degree days
to the 1st nymph emerged and the time to 50% of nymphs emerged were not affected by
any treatment, the degree days to 100% nymphs emerged were increased by 9% for those
zinnias at lowCO2/OT compared to zinnias grown at highCO2/ST (Fig. 33).
64
4.0
Zinnias
3.5
Eggs/female/day
3.0
a
2.5
b
2.0
1.5
1.0
high CO2/ST
low CO2/OT
CO2/temperature combination
Fig.31. Effects of the CO2/temperature combination on whiteflies oviposition rate (eggs
per female per day) when infested on zinnias. Plants were grown in greenhouses at either
high CO2 with sub-optimal temperature (highCO2/ST; 786 µmol·mol-1/21.9 ºC) or low
CO2 with optimal temperature (lowCO2/OT; 397 µmol·mol-1/ 25.6 ºC). Whitefly‟s eggs
were counted every three days during the adult female‟s lifespan. Error bars represent the
Standard Error of the mean. Values with the same letter are not significantly different
(P≤0.05).
1.0
Zinnias
0.8
Nynph mortality (%)
a
0.6
b
0.4
0.2
0.0
high CO2/ST
low CO2/OT
CO2/temperature combination
Fig. 32. Effects of the CO2/temperature combination on nymph mortality of whiteflies
infested in zinnias. Plants were grown in greenhouses at either high CO2 with sub-optimal
temperature (highCO2/ST; 786 µmol·mol-1/21.9 ºC) or low CO2 with optimal temperature
(lowCO2/OT; 397 µmol·mol-1/ 25.6 ºC). Whitefly‟s nymphs were circled and mortality
was recorded throughout their developmental time. Error bars represent the Standard
Error of the mean. Values with the same letter are not significantly different (P≤0.05).
65
320
Zinnias
Degree days to 100% nymph emergence
a
310
300
290
b
280
270
260
high CO2/ST
low CO2/OT
CO2/temperature combination
Fig. 33. Effects of the CO2/temperature combination on the degree days to 100%
whiteflies emergence in zinnias. Plants were grown in greenhouses at either high CO2
with sub-optimal temperature (highCO2/ST; 786 µmol·mol-1/21.9 ºC) or low CO2 with
optimal temperature (lowCO2/OT; 397 µmol·mol-1/ 25.6 ºC). Whitefly‟s nymphs were
circled and their development followed until all nymphs emerged. Error bars represent
the Standard Error of the mean. Values with the same letter are not significantly different
(P≤0.05).
66
DISCUSSION
The unique approach of comparing plants grown at elevated CO2 and sub-optimal
temperatures (highCO2/ST) with plants grown at low CO2 concentrations and optimal
temperatures (lowCO2/OT) was challenging in terms of the literature available to discuss
the findings. Therefore, the results obtained in the current study will be discussed based
on studies that have reported individual effects of CO2, temperature, fertilization and
insect treatments in addition to the results obtained in the our growth chamber study.
Furthermore, some of the results will be discussed based on studies conducted on trees
due to the lack of literature available in herbaceous crops similar to the ones in this study.
Even though trees are different in many aspects to herbaceous plants, we still believe that
general patterns and responses still hold for crops such as petunias and zinnias. In fact, it
has been mentioned that CO2 effect on biomass enhancement is similar for herbaceous
and woody species (Kirschbaum, 2011).
As mentioned before the recorded temperature corresponding to the sub-optimal
temperature was 21.9 ˚C and for the optimal temperature 25.6 ˚C. Even though, in our
study the sub-optimal temperature was closer than the optimal temperature to the one
recommended by the literature (Ball, 1991), we still tried to keep the 3 ˚C of differential
between the two temperatures at the two compartments. The experiment was conducted
during winter time but sunny days increased temperature inside the greenhouse, resulting
in higher temperatures than the initially targeted. However, Warner (2010) documented
that development of petunias had an optimal temperature of 26 ˚C, which interestingly, is
very close to what we recorded as the optimal temperature in our experiment.
67
The unchanged dry weight obtained for petunias in response to CO2 or
temperature treatment was also observed in the chambers study, we are suggesting two
hypotheses that may explain such result. The first one is that even when temperature may
cause detrimental effects on plant growth (Sage and Sharkey, 1987), the effects of CO2
enrichment on increasing plant biomass (Bazzaz, 1990) can counteract the negative
effects of sub-optimal temperatures, thereby resulting in similar dry weight. The second
hypothesis is that the reduction of 3 ºC used in this experiment was not large enough to
cause a significant difference in plant dry weight; nonetheless, 3 ºC may represent
significant savings in heating energy in greenhouses. While no effect on dry weight was
observed in petunias, an interaction between CO2/temperature combination and insect
treatments was observed for zinnias; the decreased dry weight caused by the presence of
insects at both CO2/temperature combinations indicates that regardless of the CO2 and
temperature treatments, the plants with no insect will result in heavier plants than those
infested with insects. Even in small amounts, sucking insects like whiteflies can consume
plant‟s sap that may affect plant growth. Insect consumption has been demonstrated to be
a reason for dry weight reduction (Wu and Baldwin, 2010). Additionally, due to the
insect attack, chemical signaling by the plant can be produced, directing the resources to
the production of defensive compounds instead of dry weight accumulation (Herms and
Mattson, 1992; Walling, 2000).
Contrasting with results obtained in the chamber study, the fertilization treatment
did not cause any significant change in zinnia‟s dry weight. However, the petunia‟
response to fertilization obtained is this study was consistent with the increase in dry
weight obtained for both petunias and zinnias in the growth chambers and also consistent
68
with the increases demonstrated previously by other researchers (Bazzaz, 1990;
Franzaring et al., 2008; Goverde et al., 2002; Schutz et al., 2008).While the
CO2/temperature combination did not have any effect on plant height, the 13% increase
(P≤0.0024) observed in plants treated with high fertilization, suggests that increases in
fertility can lead to taller plants, although not necessarily resulting in plants with higher
total dry weight. No differences were observed in zinnia‟s height in terms of
CO2/temperature combination or fertilization treatments. We were not able to find
published results about the decrease in plant height due to insect attack. However, the
decrease in height observed in our experiment because of the insect presence can be
directly correlated with decreases in growth mentioned in a study by (Walling, 2000). In
such report, (Walling, 2000) it was documented that plants, in order to protect themselves
from herbivores attack, respond by redirecting C and N and resources from vegetative
growth to protective mechanisms.
The increase in the number of flowers of petunias grown at the sub-optimal
temperature found in the greenhouse study was also observed in the growth chamber
study with plants grown at elevated CO2 and sub-optimal temperature. Considering the
evidence that elevated CO2 increased the number of flowers in Petunia cv. „Primetime
White Hybrid‟ found by Reekie et al. (1997), we believe that CO2 enrichment further
accentuated the increase in the number of flowers observed here at sub-optimal
temperature. However, the smaller number of flowers found on zinnias when exposed to
high CO2 in our experiment is an indication that the effects of elevated CO2 on plant
development are variable and can be different among species (Bazzaz, 1990; Reekie et
al., 1997).
69
The increase in the number of flowers observed in plants fertilized with the high
level was consistent with results obtained in zinnias at both growth chamber and
greenhouse environments; however, in the greenhouse study such effect was produced by
the interaction between fertilization and the CO2/temperature combination. A previous
study has also reported that the number of flowers is enhanced under high nutrient
conditions (Warner, 2010). The observed decrease in the number of flowers because of
the insect presence may be caused by insect tissue consumption. The mechanism of
feeding in whiteflies, even though they might not produce significant damage, can elicit
substantial changes in plant signaling and secondary metabolites (Wu and Baldwin,
2010), which we hypothesize may result in changes in plant development such as a
decrease in total number of flowers.
The reduction in the longevity of petunia flowers grown at highCO2/ST was not
observed for zinnias. This result is consistent with the reduction in flower longevity
found in potato plants grown at high atmospheric CO2 concentrations (Heineke et al.,
1999). Moreover, two studies have found that at sub-optimal temperatures the longevity
of flowers is usually shorter than at higher temperatures (Hartley et al., 1995; Park et al.,
1998). On the other hand, we concluded that zinnia flower longevity and time to flower
was less sensitive to treatments since no significant differences were observed.
Contrary to results from previous research (Reekie et al., 1997), whose found that
time to flowering was shorter in petunias grown at high CO2 than those at low CO2, we
found that time to flowering was longer by 1.2 days in petunias grown at highCO2/ST.
Since we know that low temperatures delay plant development (Reid, 1991; Sage and
Sharkey, 1987), it can be assumed that the sub-optimal temperatures to which our plants
70
were subjected caused the increase in the time to flower observed in our study. While
temperature may delay flower development, it has been demonstrated that elevated CO2
can produce a delay in time to flower as well (Reekie and Bazzaz, 1991). Similar to the
suggestion made for our growth chamber study, we recommend an evaluation of the DIF
within the same magnitude to reject the possible effect of the DIF on plant growth and
development.
The variability of the response in nutrients concentration of petunias and zinnias
to CO2, temperature, nutrition and insect treatments in this greenhouse experiment was as
variable as the response obtained in the chamber study. Several studies (Cotrufo et al.,
1998; Curtis and Wang, 1998; Goverde et al., 2002; Korner and Challa, 2003; Taub and
Wang, 2008; Yin, 2002) have reported lower concentrations of N at high CO2 than those
at ambient CO2; however, in our study the concentrations were higher in petunias grown
at highCO2/ST than those at lowCO2/OT which agrees with the concentration found by
Frantz and Ling (2011) who observed an increased concentration of N in petunias
harvested at both five and seven weeks after transplanted.
Evidence is available that neither an increase nor a decrease has been found in N
concentrations. For instance, Liu et al. (2010) reported no difference in the total N
concentration of water hyacinth (Eichhornia crassipes) to high CO2 levels. Similarly, the
N concentration remained unchanged when 2 months-old spring wheat (Triticum
aestivum) was grown at high CO2 concentrations (700 µmol·mol-1) compared to wheat
grown at ambient concentrations (350 µmol·mol-1; Vuuren et al. (1997). While no root
structure was measured in the current experiment, Vuuren et al. (1997) demonstrated
that elevated CO2 may have some implications in the root structure resulting in the
71
increase of N uptake. Therefore, we suggest that more experiments should be conducted
studying the effect of CO2 on plant root structure and nutrients uptake for petunias. This
may help explaining the increase in the N concentration under highCO2/ST conditions.
The increased N obtained in petunias and zinnias treated with high fertilization
levels was consistent with the results obtained in the chamber study; it is expected that
plants will tend to increase the uptake if there are more nutrients available in the soil,
which might be considered luxury consumption by the plant. Investigating the effects of
temperature and nutrient availability on white birch (Betula paryrifera Marsh) under
three nutrient regimes (low, intermediate and high), it was demonstrated that plants
treated with the high regime reported higher N concentration than plants grown with the
low regime (Ambebe et al., 2009).
The increased P concentrations observed in petunias grown at highCO2/ST was
consistent with the increase in P found in petunias (Frantz and Ling, 2011) and spring
wheat (Triticum aestivum cv. „Tonic‟; Vuuren et al. 1997). In the same study of Vuuren
et al. (1997), the increase in P contents was attributed to the increased root biomass
produced by plants grown at high CO2 (700 µmol·mol-1 ). In a study conducted with
cotton (Gosipiumm hirsitum L.) the concentrations of P were lower at elevated CO2
during the first year of the experiment, however during the second year, the P
concentrations were higher in cotton plants grown at elevated CO2, suggesting that high
CO2 has the potential to lead to increased P concentrations as reported in our study (Prior
et al., 1998). Based on the previous mentioned results in petunias and that no difference
was found in P concentration across any treatment in zinnias, we assume that responses
72
in P concentrations due to changes in air CO2 and air temperature are more responsive in
petunias than zinnias.
The unchanged concentration of K and Ca in response to the CO2/temperature
combination and nutrition treatment has been previously demonstrated; in an evaluation
of the effects of elevated CO2 on nutrient cycling on a sweetgum forest (Liquidambar
spp; Johnson et al. 2004) where evidence was provided that K and Ca did not significant
change across any treatment. Furthermore, our results were different from the decreased
K concentrations for petunias and zinnias, and the decreased Ca concentrations for
petunias observed in our growth chamber study (See Chapter 2). In a study conducted
with cotton (Gossypium hirsitum L.; Prior et al. 1998) found that K concentrations at
different CO2 levels were variable. They demonstrated that K concentrations in all plant
tissues were similar year after year except for leaf tissues, where K concentration at
ambient CO2 (370 µmol·mol-1) was lower than at elevated CO2 (550 µmol·mol-1) in the
first year, but the opposite the second year.
On the other hand we speculate that zinnia leaves, experienced an increase in K
concentration because of the presence of insects. We also speculate that some chemical
signal is produced as a result of the insect attack resulting in an increase of K (Wu and
Baldwin, 2010). Similar to results for petunias in this study and for Liquidambar spp in
Johnson et al. (2004) study, calcium concentrations in zinnias did not change regardless
of treatment. The increased concentration of Mg in petunias as well as the increased S
concentration observed for petunias and zinnias exposed to high fertilization was
expected, considering that the availability of nutrients in plants treated with high
fertilization may increase the uptake of these two nutrients leading to a higher
73
concentration in the leaves. Because of the predicted increase in plant growth due to the
exposure of plants to high CO2 levels, we were expecting a higher nutrient uptake thus a
higher C concentration at highCO2/ST than at low CO2/OT the opposite was found. The
decrease observed might be attributed to some negative effects of the sub-optimal
temperature in the reduction of plant metabolism resulting in lower C concentrations.
Despite the decrease in C concentration in petunias, the C/N ratios were similar
across all treatments. A metaanalysis on more than 120 publications evaluating the
effects of CO2 on plant-herbivore interactions, revealed a decrease in the C/N ratio when
plant were grown with a two fold increase of CO2 concentration (Zvereva and Kozlov,
2006). Furthermore, since the high fertilization increased the N concentration we were
expecting a decrease in the C/N ratios, however no changes were observed. C/N ratio can
also be modified by insect feeding. Even though insect survival on petunias was poor,
there was an initial infestation that might have caused N depletion by sucking plant‟s sap;
however, we are not sure if the number of insects present per plant in our study were
enough to cause such significant N reductions. Depletion of N by insects can result in
increased C/N ratios (Herms and Mattson, 1992).
No data was available to perform the statistical analysis for insects in petunias.
Some petunia plants have harmful steroidal compounds called petuniasterones (Elliger
and Waiss, 1989) that might have produced the mortality of whiteflies. For instance,
(Elliger and Waiss, 1989), conducted a study in tomato plants and reported that many
larvae of the lepidopteran (Heliothis zea Boddie) died when they were exposed to crude
extracts of petuniasterones. Also effects of management techniques might have also
affected the insect performance (See Chapter 2).
74
Oviposition is expected to be negatively affected by sub-optimal temperatures. It
was demonstrated in this study that the oviposition rate at highCO2/ST was reduced by
almost 50% when compared to the one of plants grown at lowCO2/OT. Even though due
to the nature of our experimental design we cannot separate effects of CO2 from those of
temperature, we speculate that the reduction in oviposition rate is due to the temperature
effect more than the CO2 effect. Moreover, it is well known that insects are very sensitive
to changes in temperature. For instance, Wang and Tsai (1996) studied the performance
of the silverleaf whitely (Bemisia argentifolli ) on eggplants subjected to six constant
temperatures and they concluded that besides the increased time to reach a peak of
oviposition after adult emergence, oviposition rate was lower at 20 ˚C than that at
temperatures of 25 and 27 ˚C. While it may not have the same magnitude as the effect of
sub-optimal temperature, the effect of elevated CO2 has also been reported to produce a
decrease in the growth and development of herbivores (Bezemer and Jones, 1998).
Even though whiteflies‟ mortality was measured at both highCO2/ST and
lowCO2/OT treatments, it was significantly higher at the highCO2/ST conditions. While
development of nymphs can be delayed by elevated CO2 (Brooks and Whittaker, 1999), a
more pronounced effect can be dictated by the reduction in temperature. In fact,
whiteflies development was 3-5 times longer at 15 than at 20˚C (Wang and Tsai, 1996),
which indicates that the likelihood to have mortality events increases as temperature
decreases.
In contrast with the chamber study where we observed differences in the time to
the 1st nymph emerged, the time to 50% of nymphs emerged and the time to 100%
nymphs emerged; in the greenhouse study a difference was observed until 100% of
75
nymphs reached emergence. The increase in the degree days observed in the zinnias
grown at the lowCO2/OT was an unexpected outcome and it differed from the results
obtained in the chamber study. If the technique of enriching with CO2 accompanied with
a reduction in temperature were to be implemented, the use of the low temperature will
not represent any advantage at least in terms of time to first nymph emerged and to 50%
nymph emerged. However, based on the results for this greenhouse study, at the
highCO2/ST whiteflies will take a shorter time to reach the 100% emergence than they
will at the lowCO2/OT. We cannot explain why the whiteflies took a shorter time to
develop at the sub-optimal temperature. For future work, we suggest validation of this
result since interesting effects of the CO2 on reducing time for nymph development can
be revealed.
76
CONCLUSION
Petunias
It was demonstrated in our greenhouse study that CO2 enrichment and
temperature reduction did not affected plant growth. Furthermore, an increase in the
fertilization rate from low to high resulted in higher dry weight and taller plants. Based
on the large variation in results found in the literature about the responses of plants to
different CO2, temperature, and fertility treatments, it is important to note that our results
for the greenhouse experiment were, for the most part, consistent with those obtained in
the growth chambers. The effect of elevated CO2 at sub-optimal temperatures on plant
development positively impacted the number of flowers but reduced the flower longevity
and the time to flowering.
Overall, in this experiment the effect of high CO2 at sub-optimal temperature did
have limited effect on leaf nutrient concentration. The only changes in nutrient
concentration observed were for N, Mg, and S in plants treated with high fertilization
rates. The fact that nutrients remained unchanged in plants grown at highCO2/ST is a
very promising result since we can suggest a reduction in temperature for petunia without
altering the nutrient concentration in the plant tissue. However, we need to consider that
the increase of nutrients by the high fertilization rate mentioned earlier may help to
enhance the CO2 effects. As it was recommended for the growth chamber study, the use
of a more susceptible cultivar of petunias may help to evaluate the effect of CO2 and
temperature on insect performance as well as the interaction with plant nutrition. Even
77
though the insects did not survive long in this experiment, we were still able to observe
some changes in K concentration due to the presence of insects.
Zinnias
Similar to petunias, growing zinnias at highCO2/ST produced no differences in
plant growth. However, if insects were present plants grew shorter than those plants
grown with no insects. We suggest maintaining the fertilization at optimal levels, since
no differences were observed; this will help to optimize the fertilization applications
because there is no reason to increase the fertilization rate to high levels. Nevertheless, if
we want an increase in the number of flowers, a high fertilization rate may be
recommended. The effects of elevated CO2 at suboptimal temperatures on the number of
flowers, flower longevity and time to flower in zinnias were very small. In fact, other
than a slight decrease in the number of flowers, growing zinnias at reduced temperatures
and elevated CO2 did not negatively impact either flower longevity or time to flowering.
Fewer flowers will be observed as an effect of insect pressure than plants with no insects.
Even though there were significant differences in nutrient concentrations due to
insect or fertilization treatments, only S was affected by the main effect of
CO2/temperature combination. Growing plants at those environmental settings resulted in
similar nutrient concentrations than the typical growing conditions, thus making heating
energy savings possible without affecting the nutrient concentration of zinnias. Increasing
the fertilization rate from low to high increased N and S as well as the C/N ratio.
The benefits of growing zinnias at high CO2/ST are clear and will definitely help
to improve insect management. The reduction in whiteflies oviposition and the increase
78
in nymph mortality are excellent results that may translate in less pesticide applications.
Even if considering that the 100% of nymphs took fewer degree days to emerge at high
CO2/ST, we conclude that the reduction in oviposition in addition to the increase in
mortality, will reduce the number of whiteflies emerged.
Both Crops
In general, an extension of our study can be that energy savings may be
achievable by the implementation of CO2 enrichment accompanied by a reduction of 3 ˚C
in growing temperature. The current study was an effort to provide valuable information
about a unique approach in reducing energy use while attaining plant of similar or better
quality than those obtained at standard greenhouse cultural practices. Since limited
literature is available about the effect of sucking insects on plant growth and development
as well as nutrient concentrations in plant tissues, we suggest conducting more research
on this topic. An economic analysis to determine energy savings by growing plants at
high CO2/ST will be very valuable for the floriculture greenhouse industry. Furthermore,
studies considering the effect of elevated CO2 and temperatures on water uptake and
stomata aperture in addition to nutrition and pest factors already studied here would yield
important information regarding the industry sustainability.
79
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86
APPENDIX A: Environmental settings and protocol to rear whiteflies.
87
Main plot
treatment
Replicate
1
Suboptimal
temperature
2
1
Optimal
temperature
2
Subplot
treatment
Low CO2
High CO2
Low CO2
High CO2
Low CO2
High CO2
Low CO2
High CO2
Time (Weeks)
1
2
3
4
5
6
7
8
9
10
11
12
13 Average
401.36 401.36 396.07 388.44 311.06 359.02 354.42 340.33 335.93 377.20 357.85 347.30 352.35 363.28
779.00 778.91 765.40 780.25 772.91 765.61 597.78 191.24 95.91 426.48 819.22 768.11 750.51 637.79
590.18 590.13 580.73 584.35 541.99 562.32 476.10 265.78 215.92 401.84 588.53 557.70 551.43 500.54
885.76 817.93 792.24 767.19 802.86 797.93 789.12 793.94 791.13 829.90 840.15 816.02 818.57 810.98
444.41 443.00 427.20 400.62 365.30 339.29 360.17 378.36 284.71 322.00 481.05 400.21 .
387.19
686.37 686.37 749.91 551.18 715.60 768.37 272.00 686.68 749.74 638.71 300.27 756.96 .
630.18
400.17 386.12 374.23 357.69 327.15 280.58 216.34 261.91 278.12 292.50 298.94 297.95 .
314.31
570.00 795.11 765.86 789.57 730.61 653.80 634.45 768.03 790.57 374.52 383.67 467.39 .
643.63
Table 5: Measurements of CO2 (µmol·mol-1) recorded for each replicate in the growth chamber study. Plants at the sub-optimal
temperature were harvested at the 13th week whereas at the optimal temperature at the 12th week.
88
Main plot
treatment
Suboptimal
temperature
Optimal
temperature
Replicate
1
2
1
2
Subplot
treatment
Low CO2
High CO2
Low CO2
High CO2
Low CO2
High CO2
Low CO2
High CO2
1
18.25
18.65
20.98
20.66
20.45
20.95
20.23
20.36
2
18.28
18.73
18.70
18.07
20.45
20.95
21.06
20.50
3
17.87
17.27
18.18
17.97
20.15
20.36
20.59
20.63
4
17.22
17.37
18.26
18.06
26.29
24.41
21.24
21.31
5
15.09
15.20
18.26
18.30
21.37
20.77
20.66
20.83
Time (Weeks)
6
7
8
17.26 17.87 17.92
17.42 17.76 17.91
17.99 18.38 18.17
18.31 18.71 18.52
21.12 21.19 19.76
20.40 21.08 21.39
20.47 17.23 20.68
20.66 17.25 20.71
9
17.80
17.57
17.85
18.36
20.41
20.99
20.26
18.93
10
17.37
17.43
18.02
18.49
22.47
22.41
20.98
20.90
11
18.72
18.73
18.01
18.44
20.67
21.43
21.75
21.57
12
13 Average
17.89 17.70 17.63
18.13 18.08 17.71
18.09 18.06 18.38
18.50 18.49 18.53
20.58 .
21.24
21.30 .
21.37
21.43 .
20.55
21.19 .
20.40
DIF
2.22
2.45
4.36
3.39
Table 6. Measurements of temperature (˚C) recorded for each replicate in the growth chamber study. Differential between day and
night temperatures (DIF) is presented in the last column. Plants at the sub-optimal temperature were harvested at the 13th week
whereas at the optimal temperature at the 12th week.
Main plot
treatment
Suboptimal
temperature
Optimal
temperature
Replicate
1
2
1
2
Subplot
treatment
Low CO2
High CO2
Low CO2
High CO2
Low CO2
High CO2
Low CO2
High CO2
1
22.02
23.93
23.24
24.76
23.09
27.2
21.04
23.58
2
20.82
22.91
24.35
24.55
23.25
26.98
20.42
23.64
3
22.15
24.3
24.27
24.65
19.24
26
20.68
23.74
4
21.9
23.99
24.26
24.74
24.28
27.56
18.39
20.87
5
18.82
20.29
24.22
24.57
24.57
27.24
20.61
23.04
Time (Weeks)
6
7
8
22.92 21.65 19.63
23.66 23.49 23.25
24.13 23.81 23.13
24.38 23.98 23.73
26.03 23.26 22.42
28.03 27.58 26.87
20.08 14.07 17.03
22.81 19.1 22.98
9
19.34
23.25
22.61
23.57
21.96
26.06
23.35
22.86
10
19.19
23.2
23.04
23.51
19.32
25.35
22.93
15.43
11
18.86
22.86
22.77
23.55
17.33
25.75
22.31
21.23
12
13 Average
16.92 16.42
20.05
21.14 22.71
23.00
15.03 14.58
22.26
24.22 24.2
24.19
15.41 .
21.68
25.06 .
26.64
21.84 .
20.23
20.54 .
21.65
Table 7: Measurements of light (mol·m-2·d-1) recorded for each replicate at the chamber study. Plants at the sub-optimal temperature
were harvested at the 13th week whereas at the optimal temperature at the 12th week.
89
Main plot
treatment
Suboptimal
temperature
Optimal
temperature
Replicate
1
2
1
2
Subplot
treatment
Low CO2
High CO2
Low CO2
High CO2
Low CO2
High CO2
Low CO2
High CO2
1
62.29
58.81
57.17
38.75
59.70
45.35
73.76
53.05
2
63.85
57.74
62.55
43.91
59.70
51.23
79.75
51.72
3
59.59
48.03
58.59
44.17
58.70
56.78
70.99
51.42
4
62.22
47.53
57.76
44.19
62.89
48.23
68.71
50.80
5
55.18
41.02
57.70
40.90
67.70
50.89
73.71
51.79
Time (Weeks)
6
7
8
64.05 69.14 65.84
47.28 47.28 47.34
58.05 59.05 59.90
39.32 33.07 36.92
71.32 74.54 75.67
44.64 56.72 56.89
77.74 66.03 79.60
53.18 44.27 53.08
9
69.39
47.45
60.96
42.19
89.23
55.91
68.01
57.89
10
67.22
48.12
60.65
39.37
87.92
67.85
75.74
52.85
11
65.95
44.15
59.42
35.96
84.95
58.78
75.36
51.42
12
13 Average
62.79 64.31
63.99
44.20 46.15
48.08
60.00 59.30
59.32
36.70 37.19
39.44
94.25 .
73.88
76.00 .
55.77
74.90 .
73.69
52.31 .
51.98
Table 8: Measurements of relative humidity (%) for each replicate at the chamber study. Plants at the sub-optimal temperature were
harvested at the 13th week whereas at the optimal temperature at the 12th week.
Environmental condition
Low CO2/Optimal Temperature
High CO2/ Suboptimal Temperature
Time (Weeks)
1
2
3
4
5
6
7
8
9
10
11
12
13 Average
390.1 405.0 440.5 417.8 398.0 384.8 412.3 390.5 372.1 368.3 403.6 388.8 394.4
397.4
820.9 636.3 799.4 804.9 872.6 807.6 792.0 779.4 734.4 802.6 726.3 787.6 854.7
786.0
Table 9: Measurements of CO2 (µmol·mol-1) recorded for the two compartments in the greenhouse study.
Environmental condition
Low CO2/Optimal Temperature
High CO2/ Suboptimal Temperature
Time (Weeks)
1
2
3
4
5
6
7
8
9
10
11
12
13 Average
18.89 21.46 23.13 23.58 22.21 24.25 21.83 32.13 34.24 27.13 33.72 23.95 26.86
25.64
15.47 19.35 20.24 20.37 23.23 21.03 19.79 22.87 28.37 22.61 27.30 21.44 23.84
21.99
DIF
7.54
5.77
90
Table 10: Measurements of temperature (˚C) recorded for the two compartments in the greenhouse study. Differential between day and
night temperatures (DIF) was also calculated.
Protocol to rear whiteflies (Bemisia tabaci)
The White Fly colony was started in 2004 from white flies collected from Poinsettia plants and
kept between 27 and 32.oC, x % humidity and 16:8 L:D cycle.
Materials
Poinsettias plants (Euphorbia pulcherrima) (See growing plants protocol)
100% polyester fabric
PVC frame (27” height x 27” wide x 34” length)
Plastic trays (2.5” height x 9.5” wide x 20” length)
Garbage bags
Fertilizer solution: 150 ppm 20-10-20 (181.7g/gal)
100 ppm 15-5-15 (161.5g/gal)
Room conditions: (Room 117)
Light: 16 hr of light (artificial lights from 6 a.m. to 10 p.m.)
T°: Ceiling vent opens when temperature rises over 71°F (one vent has to be opened manually)
and the artic coolers work when temperature rises over 80 °F. The thermostat starts the heat
when temperatures are below 70 °F. Refer to key points to adjust T°.
Cages:



The colony has to be kept inside a 100% polyester fabric cage framed with PVC.
Normally, four cages are used; this allows having backup cages in the case that they get
contaminated or the plants suffer any disease or other problem.
A maximum of six poinsettias have to be placed inside each cage and one dripper (at least
60 ml/min) placed per plant.
The pots have to be raised on trays placed up side down to avoid the fabric getting in
contact with the pot. Trays, fabric and PVC frames should be cleaned and disinfected
with bleach (20ml/l) every three months, also the drippers should be checked to see if
their delivery of water is adequate (it has to be measured).
Maintenance of the colony:
1. Bring 2 poinsettias plants from USDA 4 (one plant from each cage will be changed every
week).
2. Check if the plants are clean, it is important to check if the plants have no insect
contamination or other types of problems.
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3. In the colony select the plant to be discarded, for this check the rotation schedule in the
side or front of the cage (usually the oldest plant is the one to leave the cage).
4. To infest the new plant cut leaves infested with whiteflies from the discarded plant and
put them on top of the new plant.
5. When you open the cage, it is important to check:


If the drippers are working properly (60ml/min) for this you can make the
irrigation system work or touch the soil of the plants to check how moist it is.
If a dripper is not working check if it is clogged or check the timer, it is possible
that the batteries are not charged; fully charged batteries may last for a year.
6. Fertilize twice a week all the plants with the fertilizer solution that is in a bucket inside
the colony room (20-10-20 and 15-5-15).
7. Infested poinsettias plants should be kept for four weeks inside the cage.
8. In the case of contamination (plants infested with other types of insects, fungi or
diseases) the plants should be disposed earlier.
9. Plants to be disposed and fallen leaf material should be placed inside a garbage bag. To
avoid environmental and cross contamination, freeze the bags for 48 hr at -20oC before
throwing them in the trash.
Key points:

In summer (start paying attention by the end of March) close the steam valves and if
necessary open the vents on the side of the room and one on the ceiling to avoid plants
wilting or having problems with fungi. The evaporative cooler can also be used and this
can be set from low vent (spring) to high cool (summer).
 In summer the plants look wilted they may need a longer irrigation period or more
irrigation everyday. For example, in summer irrigations of 6 min everyday are used,
frequency and/or duration of irrigation should be reduced in the winter.
 In winter (start paying attention by mid October) you need to close the vents and artic
coolers because of the low temperatures, cold temperatures might kill the plants or make
the life cycles of your insects too long.
 In case of high level of contamination of other insects or plant diseases in a cage, all
plants should be discarded; clean the cage and start a new colony. The new plants should
be infested with a leaf infested with whiteflies from your non contaminated room.
 If you need to increase the colony create a new cage 1 month before the date when the
whiteflies are needed.
 Work on the colony at the end of the day to prevent contamination. Use overall and if
you need to go anywhere else remove the overall and disinfect your hands.
Room maintenance:
92


When algae grow on the floor apply greenshield (1 tbsp/gal), use the heavy duty sprayer.
Don‟t turn off the light switch in this room, because that will turn off the lighting system.
Recommendations:

Try to add a Dosatron to the irrigation line.
In case of emergency:
Follow the Laboratory Safety Guide or call:
Luis Cañas: 330-317-4102
Jim Hacker: 330-464-8889
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