The effect of the Russian wheat aphid on cold-hardiness of acclimating winter wheat seedlings
by Eric William Storlie
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in
Agronomy
Montana State University
© Copyright by Eric William Storlie (1991)
Abstract:
Affects on cold-hardiness of winter wheat, Triticum aestivum L., seedlings was investigated after an
infestation by the Russian Wheat Aphid, Diuraphis noxia (Mordvilko). Osmotic potential
measurement, carbohydrate analysis and field survival estimation were engaged as indicators of
coldhardiness. An average infestation of 147 aphids per plant, under simulated hardening conditions,
increased the osmotic potential of Froid and Brawny seedlings by 2.36 and 3.45 bars, respectively. A
natural field infestation averaging 97 aphids per ten plants, under natural hardening conditions, reduced
fructan contents of Froid and Brawny seedlings by 2.26 and 5.91 mg/100 mg dry weight, respectively;
similiar trends occurred after a natural freeze period. Field survival and yield results indicate that an
autumn infestation reduced the winter-survivability of Brawny by 54 percent and reduced grain yields
by 17.77 bushels per acre; survival and yield of infested Froid were not significantly affected. PI
372129, a resistant winter wheat line, was not affected by D. noxia in regard to these parameters.
THE EFFECT OF THE RUSSIAN WHEAT APHID
ON COLD-HARDINESS OF ACCLIMATING
WINTER WHEAT SEEDLINGS
by
Eric William Storlie
A thesis submitted in partial fulfillment
of the requirements for the degree
of
Master of Science
in
Agronomy
MONTANA STATE UNIVERSITY
Bozeman, Montana
November 1991
of a thesis submitted by
Eric William Storlie
This thesis has been read by each member of the thesis
committee and has been found to be satisfactory regarding
Approved for the Major Department
Head, Major Deplar^tment
Approved for the College of Graduate Studies
Graduate Dean
iii
STATEMENT OF PERMISSION TO USE
In presenting this thesis in partial fulfillment of the
requirements for a master's degree at Montana State
University, I agree that the Library shall make it available
to borrowers under rules of the Library.
Brief quotations
from this thesis are allowable without special permission,
provided that accurate acknowledgment of source is made.
Permission for extensive quotation from or reproduction
of this thesis may be granted by my major professor, or in
his absence, by the Dean of Libraries when,
in the opinion
of either, the proposed use of the material is for scholarly
purposes. Any copying or use of the material in this thesis
for financial gain shall not be allowed without my written
permission.
Signature
Date
iv
ACKNOWLEDGMENTS
I thank professors Luther Talbert, Hayden Ferguson,
Jarvis Brown, Greg Johnson, Jack Martin and Allan Taylor for
their advice and direction.
V
TABLE OF CONTENTS
Page
APPROVAL ...............................................
ii
STATEMENT OF PERMISSION ...............................
iii
ACKNOWLEDGEMENTS ......................................
iv
TABLE OF CONTENTS .....................................
V
LIST OF TABLES ....................
LIST OF FIGURES ...........................
ABSTRACT .........................
vii
viii
ix
I
LITERATURE R E V I E W .....................
2
MATERIALS AND METHODS .................................
9
Cultivars ...............
Growth Chamber Experiment
Experimental Design
Growing Conditions .........................
I n f e s t a t i o n ........
Hardening and Freezing ...............
Theory of Osmotic Potential Measurements ..
Osmotic Potential Measurements ............
Calibration ...........................
E s t i m a t i o n ............................
Field Experiment .................................
Experimental Design ........................
P l a n t i n g .... ..........................
Infestation .....................
Insecticide Applications..............
Sample Preparation .........................
Carbohydrate Extraction ...............
Carbohydrate Analysis ......................
HPLC ...................................
Standard Preparation .................
Sample Hydrolysis .....................
Separation Process ....................
Integration Process ..................
VO VO VO
INTRODUCTION ...........................................
10
10
10
11
12
13
14
14
14
14
15
15
15
16
17
17
17
18
21
21
vi
TABLE OF CONTENTS— Continued
Field Survival Estimation .......
Grain Yield E s t i m a t i o n .......
Statistical Analysis ....................
Page
21
22
22
RESULTS AND DISCUSSION ............................
23
Growth Chamber Experiment ........................
Diuraphis Noxia Population..................
Osmotic Potential.... . ..............
Hardening Period .................
Freeze Period .
Field Experiment .............................
Diuraphis Noxia Population .................
Fructan Analysis .................
Hardening Period .......................
Freeze Period ...........................
Sucrose Analysis ............
Hardening Period ..............
Freeze Period .................
Field Survival Estimates ....................
Grain Yield Estimates .......................
23
23
23
23
24
27
27
28
28
29
33
33
33
36
38
CONCLUSION ...................
40
LITERATURE CITED .....................
42
APPENDIX ...............
49
vii
LIST OF TABLES
Table
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Page
Mean squares for Diuraphis noxia population
counts (Growth Chamber Experiment)...........
23
Mean squares for Diuraphis noxia population
counts (Field Experiment)...............
28
Temperature and precipitation data for
1990/1991 growing seasons.....................
50
Mean squares for osmotic potential
measurements after a simulated hardening
p e r i o d .... ....................
51
Mean squares for osmotic potential
measurements after a simulated freeze
period .................. ........ . . ..........
52
Mean squares for fructan analysis after
a hardening period in the field .............
53
Mean squares for fructan analysis after
a freeze period in the field ...............
54
Mean squares for sucrose analysis after
a hardening period in the f i e l d .............
55
Mean squares for sucrose analysis after
a freeze period in the field ................
56
Mean squares for field survival estimation
after a winter s e a s o n .... ...... .............
57
Mean squares for yield estimates after a
summer growing season in the field ....
58
viii
LIST OF FIGURES
Figure
1.
Page
HPLC chromatogram of carbohydrate
standard.
Peaks are fructan (I), sucrose
(2), glucose (3), fructose (4) .........
19
HPLC chromatogram of a sample before
hydrolysis (A) and after hydrolysis (B). Peaks
are fructan (I), sucrose (2), glucose (3),
fructose (4) ...................................
20
3.
Osmotic potentials after h a r d e n i n g ......
25
4.
Osmotic potentials after freezing ............
26
5.
Fructan
contents after hardening ..............
30
6.
Fructan
contents after freezing .........
31
7.
Sucrose
contents after hardening ..............
34
8.
Sucrose
contents after freezing ..............
35
9.
Field survival estimates ......................
37
10.
Grain y i e l d s ........... ............... .......
39
2.
ABSTRACT
Affects on cold-hardiness of winter wheat, Triticum aestivum
L . , seedlings was investigated after an infestation by the
Russian Wheat Aphid, Diuraphis noxia (Mordvilko). Osmotic
potential measurement, carbohydrate analysis and field
survival estimation were engaged as indicators of cold­
hardiness.
An average infestation of 147 aphids per plant,
under simulated hardening conditions, increased the osmotic
potential of Froid and Brawny seedlings by 2.36 and 3.45 r
bars, respectively.
A natural field infestation averaging
97 aphids per ten plants, under natural hardening
conditions, reduced fructan contents of Froid and Brawny
seedlings by 2.26 and 5.91 mg/100 mg dry weight,
respectively; similiar trends occurred after a natural
freeze period.
Field survival and yield results indicate
that an autumn infestation reduced the winter-survivability
of Brawny by 54 percent and reduced grain yields by 17.77
bushels per acre; survival and yield of infested Froid were
not significantly affected.
PI 372129, a resistant winter
wheat line, was not affected by D. noxia in regard to these
parameters.
I
INTRODUCTION
The Russian Wheat Aphid, Dxuraphis noxia (Mordvilko),
was first sighted in a Montana grain field in September
198724.
Its ability to survive and proliferate in cold
climates, and its destructive feeding habits aroused concern
for Montana grain production.
A possible effect after an
autumn D. noxia infestation on winter grain seedlings may be
a reduction of cold-hardiness.
This thesis sought to
determine if the winter-survivability of three winter wheat
types - Froid, Brawny and PI 372129 - may be affected by an
infestation.
Winter-survival is a pertinent concern for
grain producers in Montana, since winterkill is a potential
outcome for most winter grains.
Death is typically
attributed to an inherent vulnerability of plants.
If
winterkill is partly attributable to a D. noxia infestation,
the problem may be partly remedied with control strategies
during autumn or with resistant winter whe a t .
2
LITERATURE REVIEW
The ability of Diuraphis noxia to survive on winter grain
seedlings through fall acclimation periods in Montana has
prompted concern for its effects on winter wheat cold­
hardiness.
Survival and natality studies suggest that D.
noxia survived temperatures between -4° and -2 S0C5 and bore
nymphs at least to 5°C36.
D. noxia adjusts reproduction
rates and generation periods according to temperature and
food availability - reproduction rates decrease and
generation periods increase when temperatures are low and
small grain growth is minimal1,35,36.
Diuraphis noxia putatively injects a toxin while
probing plant tissues for food. Symptoms which lead to a
toxin-theory are intervenal chlorotic yellow and purple
streaks on the leaf surface and organelle damage at the
ultrastructural level13; analysis of chloroplasts from
wheat, Triticum aestivum L . , tissue treated with an aphid
extract revealed chloroplast membrane disintegration13.
Thus, implications of an infestation are not only a loss of
carbohydrates from ingestion but also irreversible damage to
the organelles necessary for sugar production.
Since soluble carbohydrates have been associated with
3
winterhardiness in small grains2,41,45, the question of a D.
noxia influence on the hardiness of winter grains may be
pertinent.
There are several approaches for testing the
coldhardiness of winter grains.
Fowler et al.14 evaluated
34 possible tests and correlated some tests with a Field
Survival Index (FSI).
The tests most highly correlated with
FSI were LT50, tissue water content, plant erectness, crown
phosphorus content and total sugar content.
DeNoma et al.9
indicate a linear relationship existed between crown osmotic
potential and field survival scores for five winter wheat
varieties during two winter seasons.
Lower osmotic
potentials of crown tissue were associated with increased
winterhardiness.
The results of Thomas and Taylor49
corroborate the results of DeNoma et al. with a study that
suggests a more winterhardy variety, Froid, had a lower
osmotic potential than a less winterhardy variety. Brawny,
after a freeze period.
When the cell-dessication theory of freeze-injury is
considered, the results of DeNoma et al.9 may correspond
with the negative correlation between crown moisture and FSI
observed by Fowler et al.14
Since freeze-injury is believed
by some to be a result of intracellular dehydration, a
mechanism of hardiness may involve osmoregulation.
By
increasing its osmotic concentration, a cell may achieve
vapor pressure equilibrium with the extracellular
4
environment during cold periods when the extracellular
solution may freeze27,46.
In regard to this theory, a more
negative osmotic potential may be explained by an increase
in solutes and proportionate decrease in moisture content,
which would prevent plasmolysis and mitigate the
concentration of toxic solutes.
Osmoregulation is, at least in part, a function of
soluble carbohydrate concentrations17,27,39.
A compelling
candidate for a carbohydrate that can adjust the osmotic
potential of a cell is fructan, a storage polymer of
fructose which is found in at least 36,000 species
representing ten families of plants7.
A fructan molecule
consists of two or more (2->6 or 2->l)-B-D-fructofuranans
bound to one D-glucopyranose residue.
Molecules may exist
as linear pleins, with (2->6) linkages; inulins, with (2->l)
linkages; or branched, with (2->6 and 2->l) linkages7,15,33,40.
Fructans of wheat consist mostly of plein backbone linkages
with (2->l and 2->6)
linked, branch residues7.
Suzuki and Nass47 enumerated the advantages of fructan
over starch as a cold temperature storage carbohydrate:
fructan has high solubility in water, it is resistant to
crystallization at subzero temperatures, and fructan
biosynthetic pathways are insensitive to low temperatures.
The advantages of fructan, however, do not suggest that
starch is replaced in cool-season grasses.
Chatterton et
al.8 defined fructan as an ancillary form of carbohydrate
5
storage; its accumulation in the vacuole allows
photosynthesis to continue in leaves during cool periods
when other storage pools are saturated.
Two enzymes are implicated in fructan synthesis in
cereals: sucrose: sucrose fructosyl-transferase (SST) has
been investigated in small grains22'23,50'53; fructan: fructan
fructosyl-transferase (FFT) has not been investigated in
small grains.
SST transfers a fructose moiety from one
sucrose molecule to another, producing a ketose and a
glucose molecule53.
In Jerusalem artichoke, Helianthus
tuberosus L . , FFT transfers single terminal B-Dfructofuranosyl residues to the same position of another
fructan molecule11,12.
A putative FFT in small grains is
speculated to perform an analagous role as in H.
tuberosus53.
Depolymerization of fructan molecules is accomplished
by the activity of fructan hydrolase, according to studies
on H. tuberosus, Festuca arundinacea (Schreb.) and T.
aestivizm12,25'43'50.
Tognetti et al.50 and Jeong and Housley23
suggested that fructan hydrolase activity decreased during
cold acclimation and subsequently increased during warm
acclimation in whe a t .
The role of fructans in osmoregulation is conceivable
in consideration of the fructan-specific enzymes and the
capability of fructan polymers to exist in various degrees
of polymerization (DP). FFT may redistribute fructose
6
residues from a larger polymer to a smaller one and
consequently increase the concentration of lower molecular
weight fructans.
This mechanism allows for rapid changes in
solute concentrations of cells and consequently influences
the osmotic potential40.
A high Km value for SST suggests sucrose must
accumulate to a relatively high concentration before fructan
is synthesized and begins to accumulate6,19.
Pollock38
provides evidence from a study on Lolium temulentum L. that
sucrose concentrations rapidly increased before fructan
accumulated. Bancal and Gaudillere2 and Wagner et al.53 show
similiar results in T. aestivum.
Similiarly, Labhart et
al.25 conclude that a threshold level of low DP fructans and
sugars were necessary for the synthesis of high DP fructans
in Festuca pratensis (Huds.).
Chatterton et al.8 evaluated fructan accumulation in
185 Gramineae accessions and suggests that total
nonstructuraI carbohydrate (TNC) accumulation was more
closely related to fructan accumulation than sucrose; a
threshold value of 15% TNC was postulated as necessary for
fructan accumulation.
Livingston et al.29 present results
from a study on Hordeum vulgare L. that corroborate the
results of Chatterton et al.8.
In any case, it appears
fructan accumulation is induced by a changed ratio of carbon
utilization to carbon fixation41.
Several studies on the relationship between fructan
7
accumulation and cold-hardiness of winter cereals suggest
that significant correlations existed between fructan
content and hardiness30,47,49,51'57.
Tognetti et al.51 indicate
an association occurred between hardiness in winter wheat
and sucrose and fructan accumulation at 40C.
Livingston30
analyzed the accumulation of each DP fructan in Avena sativa
Ti., H. vulgare, T. aestivum and Secale cereale L. after a
20C hardening period.
Results of this study suggest that
the least winterhardy cereal, A. sativa, accumulated as much
sucrose and DP3-DP7 as the other, hardier species.
However,
A. sativa accumulated significantly less DP>9 fructan than
the other cereals30.
Suzuki and Nass47 present similiar
results and suggest that high DP fructans were more closely
associated with hardiness than low DP fructans.
Thomas and Taylor49 studied the relationship between
crown osmotic potential, fructan content and winterhardiness
in Froid and Brawny winter whe a t .
After a simulated
hardening period, their results indicate that Froid, the
more winterhardy variety, accumulated more fructans and had
a lower osmotic potential than Brawny, the less winterhardy
variety.
Furthermore their results suggest a negative
correlation occurred between fructan content and osmotic
potential for Froid and Brawny, after a hardening period.
The negative correlation of this study provides evidence for
the role of fructan in osmoregulation.
8
Though the mechanisms of fructan involvement in freezeresistance remain tenuous51, an association between fructan
accumulation, cold temperatures and relative cold hardiness
seem apparent in consideration of the above cited research.
Several investigations used fructan levels to infer effects
on winterhardiness caused by pests21'31,54'55,56.
Holmes et al.21
suggest that a greenbug, Schizaphis graminum (Rondani),
infestation in T. aestivum reduced the level of high DP
fructans and may conseguently have reduced freezing
resistance. Livingston and Gildow31 studied affects of
Barley yellow dwarf virus on fructan concentrations in A.
sativa crowns and suggest that fructan levels of infected
oats were significantly less than the controls.
9
MATERIALS AND METHODS
Cultivars
The winter wheat types used in the growth chamber and
field experiments were Froid, with a high winterhardy score
of 4.5 (on a scale of I to 5)9; Brawny, with a low
winterhardy score of 2.09, and PI 372129, a D. noxia
resistant accession with an undocumented hardiness42.
Growth Chamber Experiment
Experimental Design
The growth chamber experiment provided plant samples
for osmotic potential measurement. It was designed as a
split-plot randomized complete block with four blocks.
Each
plot either was infested with D. noxia or was not infested
(control), and each subplot consisted of 28 subsamples of
one winter wheat type.
Four subsamples were used to measure
the osmotic potential for a hardening and a freezing period
(N = 96).
The primary interest in this study was a
comparison of the osmotic potential for the infested and
control treatments of each variety (treatment combinations).
Seeds for each of the samples were planted two cm deep
in sand-filled, 1.5" diameter cone-taihers (Supercell Cone-
10
tainer).
Two blocks were grown at a time in a customized
environmental growth chamber for the duration of a growing
and hardening period.
Growing Conditions
For the first 33 days of growth, temperatures were
16°C during the 12 hour light period and 14°C during the 12
hour dark period.
Light was emitted from four 400 watt,
multivapor and four 400 watt, luclox lamps (General
Electric) with a photosynthetic-photon-flux-density of 1200
jLtmol/m2 min.
Plants were watered daily, oh alternate days
with HoaglandzS full strength nutrient solution.
Infestation. Ten days after planting, when seedlings
consisted of two leaves, the infested plots were inoculated
with three aphids per plant using a damp artist's paint
brush.
Cages for confining the aphids were made of veil
fabric on the sides and a plexiglas cover.
The final aphid
population per plant was determined from counts of five
individual plants of each variety from the four blocks (N =
60) .
Hardening And Freezing. The following hardening and
freezing procedures were adapted from Gullord et al.18.
The
hardening period began 33 days after planting and entailed
reducing growth chamber temperatures to a continuous 4°C
setting.
Additionally, the luclox lamps were switched off
and the multivapor lamps set for continuous lighting with a
photosynthetic-photon-flux-density of 280 jumol/m2 min.
11
These conditions were maintained for 21 days.
After 21 days of hardening, four subsamples from each
subplot were removed and cut to a one cm, soil-free crown
section.
The crown was immediately placed into a I ml
microcentrifuge tube and froze in liquid nitrogen for two
minutes and subsequently stored at -7O0C.
Concurrently,
four subsamples from each subplot were prepared for a freeze
period.
Plants were cut to 5 cm sections - including root,
crown and leaf tissue.
These sections were immediately
placed into slots of a moist cellulose sponge which was
pressed onto a nipple and flange.
Samples were placed into
a Lo-Cold freezer (ScienTemp) with no light and a
temperature of -2°C for a 56 hour prefreeze.
The
temperature was monitored with a Thermocouple Thermometer
(Omega); five thermocouple wires were randomly distributed
to sites adjacent to crowns.
After the -2°C prefreeze,
temperatures were reduced 2° per hour until -V0C was
reached.
Samples were removed after two hours at this
temperature and immediately cut to I cm crown sections,
placed in I ml microcentrifuge tubes and froze in liquid
nitrogen for two minutes before storing at -7O0C.
Theory Of Osmotic Potential
Measurements
Theoretically, the freezing and eventual thawing of
plant tissue results in disruption of cellular membranes; a
process that prevents the generation of turgor, so only the
12
energy of osmotic potential is measured3. The components of
water potential are described by the following simplifified
equation3:
Vv-^o+Vp
where o and p represent osmotic and pressure forces,
respectively.
When the pressure component is canceled by
freezing and thawing, the equation reduces to two factors3:
Therefore, measurement of water potential approximates the
osmotic potential.
Osmotic Potential Measurements
While frozen, crown samples were transferred to 1.1
cubic cm sample and calibration chambers; chambers were
immediately fastened to Leaf In-situ thermocouple
psychrometers (J.R.D. Merrill).
Depth of the psychrometer
end window was adjusted to avoid contact with the crown
tissue.
The psychrometer and 30 cm of lead wire were
immersed in a 25°C water bath for a two hour equilibration
period; this period facilitates vapor pressure equilibrium,
when the water potential of the tissue and vapor pressure of
the chamber are in dynamic equilibrium4.
The water bath
prevents severe temperature gradients that may cause water
13
vapor to migrate from warmer to cooler sectors of the
chamber4.
After equilibration, a Viking connector (J.R.D.
Merrill) at the terminus of the lead wire was connected to a
HR-33 microvoltmeter (Wescor).
A current of about 5 ma was
transmitted through the psychrometer unit for 30 seconds.
The current was passed through the lead wires to a chromeI
and constantan sensing junction, and the Peltier effect
cooled the junction slightly below the dewpoint temperature
of the enclosed atmosphere52. This resulted in water
condensing on the sensing junction.
When the current was
terminated, the condensed water evaporated at a rate that
was a function of the equilibrium vapor presure52.
Evaporative cooling reduced the temperature of the sensing
junction, and the difference in temperature between the
reference and sensing junctions produced an electromotive
force (emf) output which was registered by the
microvoltmeter52.
Calibration. Lang26 documented the relationship between
water potentials and NaCl solutions at temperatures between
O0 and 4O0C.
Based on this relationship, calibrating each
of the thermocouples involved measuring four concentrations
of NaCl - 0.30, 0.50, 0.60 and 0.80 molal - on filter paper
at an equilibration temperature of 25°C.
Calibration curves
of the microvolt outputs vs. waterpotential,
plotted.
in bars, were
Finally, regression equations were computed on
14
MSUSTAT32 to express the curvilinear relationship between
microvolt output and water potential for each thermocouple
psychrometer.
Estimation Theory. The method of estimating osmotic
potentials is an inference from the vapor pressure produced
by crown tissue in the sample and calibration chamber.
Vapor pressure is quantitatively related to the water
potential by the following equation:
Vw-
where R is a gas constant, T is temperature (Kelvin), Vw is
partial molal volume of water (m3 mol'1) , ew is vapor
pressure and e0 is saturated vapor pressure.
Therefore,
estimates of ew will provide estimates of water potential if
the temperature is fixed and a thermodynamic equilibrium
exists, since all factors other than ew will remain constant
under these conditions3.
Field Experiment
Experimental Design
Planting. The field experiment was planted at the Fort
Ellis research plots on September 5, 1990.
It provided
winter wheat samples for carbohydrate analysis, field
survival estimation and grain yield estimation. This
15
experiment was designed as a split-plot randomized complete
block with four blocks.
Each plot was either infested with
D. noxia or was sprayed with insecticides (controlled); each
subplot consisted of six rows of one variety, planted at a
density of 15-20 seeds per square foot for ten foot rows.
There was one sample measurement for each of the subplots
for each parameter (N = 24). The primary interest in this
study was a comparison of infested and controlled treatments
of each variety (treatment combinations).
Infestation. Infested plots were naturally infested
with D. noxia.
Populations per ten plants were estimated on
September 30 by counting aphids on ten randomly selected
plants from each subplot (N = 24).
Insecticide Applications. The controlled plots were
sprayed with Asana (EI-Dupont) at a rate of 0.035 pound
active ingredient per acre on September 19; Lorsban
(DowElanco) was applied at a rate of 0.50 pound active
ingredient per acre on September 27.
Sample Preparation
On October 30, after a hardening period (Table 3,
Appendix), plants were randomly selected from the second and
/
fifth rows of each subplot and immediately trimmed to I cm
crown sections.
The crowns were immersed in liquid nitrogen
and stored on dry ice until the samples could be lyophilized
in a freeze-drier (Virtis) for 24 hours. On December 10,
after a freeze period (Table 3, Appendix), a second
16
sampling was removed from the frozen soil with a pick-hammer
and processed the same as the preceeding samples.
Carbohydrate Extractio n . The dried crown samples were
ground to a powder with a mortar and pestle and sifted
through a 30 mesh tea strainer.
Samples of 50 mg were
weighed for carbohydrate extraction.
placed in a 5 ml plastic test tube.
Each 50 mg sample was
As adapted from Smith
and Grotelueschen 44, three ml of 80% ethanol was added to
each sample, vortexed and placed in a VO0C water bath with
vigorous agitation.
After 15 minutes of agitation, the
tissue had settled sufficiently to allow decanting without
centrifuging.
The ethanol extraction was repeated another
time before replacing ethanol with distilled, deionized
(ddi) water for three cycles.
The 80% ethanol extracts
glucose, fructose, sucrose and lower DP fructans from the
tissue, and water at VO0C extracts all fructans as well as
sucrose, glucose and fructose44.
Sample preparation and High Performance Liquid
Chromatography (HPLC) methods were adapted from Livingston
et al.29.
Supernatants from the extractions were passed
through a column of 4cc Amberlite MB-3A (Aldrich)
ion-
exchange resin, and the column flushed with 20 mis ddi water
into 30 ml beakers.
All beakers were placed in a GO0C
evaporating oven until each sample was completely
evaporated.
A micropipette was used to add 1.25 mis of ddi
water to each beaker to resuspend the carbohydrates and to
17
transfer the solutions into 1.50 ml microcentrifuge tubes.
Samples were centrifuged at 11,750 x g for 15 minutes.
The
supernatant was decanted with a micropipette and transferred
to a new microcentrifuge tube.
Samples were vacuum dried in
a Speedvac Concentrator (Savant) until completely
dehydrated.
Subsequently, samples were resuspended in ddi
water to produce a I ml solution.
These carbohydrate
solutions were stored at -2O0C.
Carbohydrate Analysis
High Performance Liquid Chromatography. The HPLC system
was composed of a Spectroflow 400 pump (Kratos), an injector
(Rheodyne) with a 20 ul sample loop, a Carbo-C guard column
(BioRad), a HPX-42C carbohydrate column (BioRad), a
Differential R401 refractometer (Waters), and a Data Module
M730 integrator (Waters).
The column was heated to 85°C
using a water jacket and water bath.
Standard Preparation. Carbohydrate standards for
sucrose, glucose and fructose were prepared from reagentgrade, commercial stocks.
A fructan standard was prepared
from a dried, ground mixture of crowns from cold-hardened
Froid, Brawny and PI 372129.
The ethanol and water
extraction procedure was used to concentrate fructans from
fifteen 100 mg samples.
After the extraction, solvents were
evaporated from 30 ml beakers in a 65°C oven.
Subsequently,
carbohydrates in each beaker were concentrated into a 1.25
ml solution by successively resuspending each sample with
18
an original 1.25 ml ddi water.
The 1.25 ml carbohydrate
solution was centrifuged and vacuum dried as previously
described for the sample-extraction procedure.
precipitate was visible at this stage.
A white
The precipitate was
washed with 500 /zl 80% ethanol and centrifuged 2 minutes at
16,000 x g.
The supernatant was discarded and the
precipitate washed two more times with 500 /il of 80% ethanol
to remove all detectable glucose and fructose and most
sucrose.
Any remaining detectable sucrose was calculated on
the HPLC with a sucrose standard and factored into the final
sucrose standard.
Some of the lower, 3-5 DP fructans were
removed by the ethanol.
However, all DP fructans were
grouped as one fraction for quantification, and the most
prominent fraction, DP>6, was fully retained during the
extraction process (Figure I ) .
A 5 ml carbohydrate standard
was prepared With 50 mg fructan, ISmg sucrose, 10 mg glucose
and 10 mg fructose for calibrating the integrator each day
before a series of sample injections.
Sample Hydrolysis. To confirm that the observed
fructan peaks were polymers containing a terminal glucose
residue linked to various DPs of fructose residues, 500 //I
of a carbohydrate solution, extracted from a hardened
Brawny, was hydrolyzed with 500 ,jLtl of 0.3 molar HCl for two
hours at 8O0C.
Then the sample was neutralized with 0.3
molar NaOH and passed through an ion-exchange resin.
resin was flushed with 25 ml of ddi water into a 30 ml
The
19
Figure I.
HPLC chromatogram of carbohydrate standard.
Peaks are fructan (I), sucrose (2), glucose
(3), fructose (4).
beaker with the hydrolyzed sample. Subsequently the solvents
were completely evaporated in a 6O0C oven and the sample
carbohydrates resuspended to a 500 /il solution with ddi
water.
The solution was filtered through a 0.45 micron
filter before injection into the HPL C .
Hydrolysis was not
complete, though a comparison of the ratio of concentrations
between hydrolyzed and nonhydrolyzed samples revealed that
the putative fructan peaks were composed of glucose and
fructose residues, with a higher proportion of fructose
(Figure 2).
The ratio of glucose to fructose was 1:2 before
hydrolysis and 1:3 after hydrolysis.
20
Figure 2.
HPLC chromatogram of a sample before hydrolysis
(A) and after hydrolysis (B). Peaks are fructan
(I), sucrose (2), glucose (3), fructose (4).
21
Separation Process. Degassed, ddi water was used as the
solvent for HPLC operation.
Twenty jul of each carbohydrate
sample was injected for each analysis.
Once injected, the
sample entered a solvent stream which flowed at 0.5 ml per
minute.
It passed through a precolumn that retained
contaminants, preventing them from binding to the
carbohydrate column.
The sample was then separated into
individual carbohydrate fractions by processes of size
exclusion and ligand exchange.
primarily separated by size.
Fructans and sucrose are
The monosaccharides are
separated primarily by ligand exchange; a sampIe-counterion
complex is formed between the carbohydrate and Ca counterion
on the resin bed34.
Integration Process. After separation, the carbohydrate
fractions passed through the detector cell of the
refractometer; a change in the refractive index caused a
deflection of the light beam that passed through the cell,
generating an ouput signal34.
The signal was received by
the integrator and translated into chromatographic peaks and
retention times for quantification and identification of the
carbohydrates.
Field Survival Estimation
The two center rows of each subplot were used to count
wheat seedlings within six, one foot row sections marked by
wire flags.
Seedling counts were made in late September,
1990 and again in early May, 1991 to determine the survival
22
percentage for each sample (N = 144).
Grain Yield Estimation
For yield estimates, the same two center rows used for
survival estimates were trimmed to eight foot rows and cut
with sickles for threshing in a Wintersteiger combine.
Weights were extrapolated to bushels per acre for each
sample (N = 24).
Statistical Analysis
Analysis of variance was computed using MSUSTAT32, and
whole and subplot error terms were used to calculate Least
Significant Difference values at the .05 level of
significance (LSD 05) for treatment combination means (Tables
4-11, Appendix). The LSD 05 equation for differences between
subplot treatments for different main plot treatments is
described by Little and Hills28.
23
RESULTS AND DISCUSSION
Growth Chamber Experiment
Diuraphis noxia Population
Population levels in the infested plots after a
hardening period were 147 aphids per plant with no
significant differences between varieties (Table I ) .
Table I.
Mean squares for Diuraphis noxia population counts
of three winter wheat types.
source
df
mean souare
n-value
block
3
33553.OO
.0014
variety
2
3547.50
.5326
blk*var
6
6719.10
.3181
48
5557.50
error
'
Osmotic Potentials
Hardening Period. After a 40C hardening period,
infested Froid and Brawny had significantly higher osmotic
potentials than the respective controls (LS D 05 = 2.12).
Infested PI 372129 was not different from the control PI.
For Froid, the control measured -25.58 bars and the infested
measured -23.22 bars.
For Brawny, the control measured
-22.76 bars and the infested measured -19.31 bars.
PI
24
372129 measured -22.46 bars (Figure 3).
These results suggest infested Froid and Brawny
accumulated fewer solutes in the crown.
The lower osmotic
potentials could also suggest the presence of more water in
the infested crowns, though there is no reason to believe
this was an effect of D. noxia.
According to DeNoma et
al.9, osmotic potentials correlated with cold-hardiness
levels - the lower an osmotic potential, the more cold-hardy
was the winter wheat.
Furthermore, Thomas and Taylor49
indicated the more cold-hardy, variety, Froid, had a lower
osmotic potential than the less hardy variety. Brawny.
The
same difference between Froid and Brawny occurred in this
investigation.
One may infer from the groundwork
established by DeNoma et al.9 and Thomas and Taylor49 that
infested Froid and Brawny were less cold-hardy than the
controls, under the 40C hardening temperature of the growth
chamber.
Freeze Period. After a -6°C freeze period,
infested
Brawny had a significantly higher osmotic potential than the
control
(LSD 05 = 2.97) .
Infested Froid and PI 372129 were
not different from the respective controls.
For Brawny, the
control measured -24.68 bars and the infested measured
-20.80 bars.
Froid measured -25.88 bars and PI 372129
measured -22.97 bars (Figure 4).
When these results are compared with results of the
hardening period, the most prominent change occurred with
25
to -I
Oi CT
treatment
Figure
3.
O s m o t i c p o t e n t i a l s a f t e r 40C h a r d e n i n g pe r i o d
(* = s i g n i f i c a n t at .05 level)
26
CO-I COD’
treatment
t
F i g u r e 4.
O s m o t i c p o t e n t i a l s a f t e r - I 0C f r e e z e pe r i o d
(* = s i g n i f i c a n t at .05 level)
27
infested Froid which became more negative and no different
from the control.
The expectation was that the osmotic
potentials for all treatment combinations would decrease
after a freeze period.
DeNoma et al.9 and Thomas and
Taylor49 indicated that osmotic potentials decreased after a
freeze to -20C, independent of cold-hardiness levels.
A decreased osmotic potential after a freeze may be
postulated as a cold survival mechanism.
Part of this
mechanism would, hypothetically, entail the breakdown of
fructans into smaller polymers and fructose molecules.
These changes would translate into a higher concentration of
solutes and consequently a more negative osmotic potential.
The -6°C freeze period did not induce an observable change
of osmotic potential for any of the treatment combinations
other than infested Froid.
The inconsistency of these
results suggest the experimental method for simulating a freeze was flawed.
Indeed the plants were subjected to
abnormal conditions using the sponge and flange materials
engaged by Gullord et al.18, and furthermore, the prefreeze
and freeze periods may have been too short to induce
detectable osmotic potential changes.
Field Experiment
Diuraphis noxia population
Population levels in the infested and controlled plots
on September 30, 1990 were 9.7 and 0.30 aphids per ten
28
plants, respectively, with no significant differences
between winter wheat types for the infested or controlled
treatments (Table 2) .
Table 2.
Mean squares for Diuraphis noxia population
counts on three winter wheat types in infested
and controlled plots.
source
df
mean souare
o-value
block
3
76.37
treatment
I
52734.00
whole plot
error
3
36.70
variety
2
228.04
.1693
trt*var
2
256.63
.1401
12
110.33
subplot error
.0000
Fructan Analysis
Hardening Period. After a hardening period in the
field, infested Froid and Brawny had a significantly lower
fructan content than the respective controlled treatments
(LSD 05 = 1.86).
Infested PI 372129 was not different from
the controlled treatment.
For Froid, the controlled
treatment measured 32.27 mg fructan per 100 mg dry weight
and the infested measured 30.01 mg.
For Brawny, the
controlled treatment measured 25.94 mg fructan and the
infested measured 20.03 mg.
PI 372129 measured 25.77 mg
29
fructan (Figure 5).
In agreement with other studies discussed in the
Literature Review47,49,51'57, more fructans accumulated in the
more cold-hardy variety than the less hardy variety after a
hardening period.
Furthermore, these results correspond
with the osmotic potentials measured after hardening in the
growth chamber; both results implying a diminished cold­
hardiness for infested Froid and Brawny.
In the light of
speculation discussed in the Literature Review on the
potential role of fructans in osmoregulation,
it appears
that increasing fructan contents correlate with decreasing
osmotic potentials.
Thomas and Taylor49 analyzed the
relationship of fructan levels and osmotic potential and
indicate that fructans were associated with 44% of osmotic
potential variation (R2 = 0.44).
Freeze Period. After a field freeze period, infested
Froid and Brawny had significantly less fructan than the
respective controlled treatments (LSD 05 = 2.58).
Infested
PI 372129 was not different from the controlled treatment.
For Froid, the controlled treatment measured 20.22 mg
fructan per 100 mg dry weight and the infested measured
17.46 mg fructan.
For Brawny, the controlled treatment
measured 11.82 mg fructan and the infested measured 7.26 mg
fructan.
PI 372129 measured 5.97 mg fructan (Figure 6).
When these results are compared with fructan contents
of the hardening period, the same differences between
30
Froid
Y//A Brawny
c - control
F i g u r e 5.
Pl
I- Infested
F r u c t a n c o n t e n t s a f t e r au t u m n a c c l i m a t i o n p e r i o d
in th e f ield (* = s i g n i f i c a n t at .05 level)
31
25
O
i
0
I
O
i
treatment
Froid
Y//A Brawny
c - control
F i g u r e 6.
Pl
I - infested
F r u c t a n c o n t e n t s a f t e r w i n t e r f r e e z e p e r i o d in
t h e f ield (* = s i g n i f i c a n t at .05 level)
32
treatment combinations are present.
The only difference
after a freeze period is a decrease in fructan contents for
each treatment combination.
The results of Thomas and
Taylor49 also suggest that fructans decreased after a -2°C
period.
The sucrose analysis suggests an opposite trend:
sucrose contents increased after a freeze period (Figures 7
and 8).
These inverse changes in fructan and sucrose
contents after a freeze period are further evidence that
fructan is involved in osmoregulation; a breakdown of larger
DP fructans to smaller molecules would increase the
concentration of solutes, decrease the osmotic potential and
consequently reduce injuries that may result from
desiccation. If indeed fructan molecules are hydrolyzed to
smaller molecules - including sucrose, glucose and
fructose - during a freeze period, then it would appear the
induction of fructan synthesis is dependent upon mechanisms
other than sucrose or TNC concentrations as hypothesized by
Pollock and others2,8,29,38,53.
The extent of cold-hardiness reduction is not possible
to infer from an analysis of fructan content.
However, a
more revealing investigation than the current thesis may
analyze individual DP fructans for more insight into the
cold-hardy status of a plant, since higher DP fructans may
be more closely associated with cold-hardiness than total
fructans30,47.
33
Sucrose Analysis
Hardening Period. After the field hardening period,
infested Froid, Brawny and PI 372129 had significantly lower
sucrose contents than the respective controlled treatments
(LSD 05 = 0.46).
For Froid, the controlled treatment
measured 4.09 mg sucrose per 100 mg dry weight and the
infested measured 3.20 mg sucrose. For Brawny, the
controlled treatment measured 4.68 mg sucrose and the
infested measured 3.03 mg sucrose.
For PI 372129, the
controlled treatment measured 4.66 mg sucrose and the
infested measured 4.00 mg sucrose (Figure 7).
Freeze Period.
After the field freeze period, infested
Froid, Brawny and PI 372129 had significantly lower sucrose
contents than the respective controlled treatments (LSD05 =.
0.41).
For Froid, the controlled treatment measured 6.73 mg
sucrose per 100 mg dry weight and the infested measured 6.10
mg sucrose.
For Brawny, the controlled treatment measured
6.86 mg sucrose and the infested measured 5.23 mg sucrose.
For PI 372129, The controlled treatment measured 5.93 mg
sucrose and the infested measured 5.43 mg sucrose (Figure
8).
There are no correlative studies that associate sucrose
with cold-hardiness, though previous studies recognize that
sucrose levels increase when T. aestivum or Lolium
temulentum L. are subjected to 40-50C38'53.
Results of this
study suggest sucrose contents also increase after a freeze
34
Frold
Y//A Brawny
c - control
F i g u r e 7.
H S Pl
I - infested
S u c r o s e c o n te nts a f t e r au t u m n a c c l i m a t i o n p e r i o d
in the field (* = s i g n i f i c a n t at .05 level)
35
Froid
Y//A Brawny
c - control
F i g u r e 8.
Pl
* ~ infested
S u c r o s e c o n t e n t s a fter freeze p e r i o d in the
f ield (* = s i g n i f i c a n t at .05 level)
36
period.
After the hardening and freeze periods, infested PI
372129 contained less sucrose than the controlled treatment,
which suggests that PI was partially susceptible to the
effects of D. noxia but not to an extent that it interfered
with fructan accumulation.
The diminished sucrose contents
of each infested wheat type may corroborate speculation
about the negative effect D. noxia has on sucrose synthesis
by means of its putative disruption of membranes.
Field Survival Estimates
Survival percentages were determined in May 1991.
Infested Brawny had a significantly lower survival
percentage than the controlled treatment ( L S D og = 25.15).
Infested Froid and PI 372129 were not different from the
respective controlled treatments.
For Brawny, the
controlled treatment measured 84.30 percent survival and the
infested measured 38.90 percent.
Froid measured 93.00
percent survival and PI 372129 measured 50.00 percent
survival
(Figure 9).
These results suggest a fall D. noxia infestation
reduced the cold-hardiness of Brawny but had no significant
affect on the hardiness of Froid or PI 372129.
In part
these results correspond with inferences derived from the
osmotic potential and fructan results: an increased osmotic
potential and reduced fructan content imply a reduced
winter-survivability for infested Brawny.
This trend is not
37
09 3 *- > — > CO
Frold
c - control
F i g u r e 9.
Brawny
Pl
I- Infested
Field survival percentages after a winter
season (* = significant at .05 level)
38
detected from the results of infested Froid, perhaps due to
its higher level of hardiness.
Thomas and Butts48 suggest that a D. noxia infestation
increased the LT 50 of Norstar winter wheat by 1.6° to 6.5°C.
If the LT 50 of infested winter wheat in this thesis was also
increased a few degrees, then it is possible the winter of
1990/1991 was harsh enough to affect an infested Brawny
without significantly affecting an infested Froid, which
would imply a semblance of resistance is imparted by
increased cold-hardiness.
Grain Yield Estimates
Grain yields were determined in August 1991.
Infested
Brawny had a significantly lower grain yield than the
controlled treatment (LSD 05 = 17.22).
Infested Froid and PI
372129 were not different from the respective controlled
treatments.
For Brawny, the controlled treatment measured
61.52 bushels per acre and the infested measured 43.75
bushels.
Froid measured 60.42 bushels and PI 372129
measured 43.53 bushels (Figure 10).
Since there was no D. noxia infestation during the
growing season, these results suggest that the spring
survival reduction of infested Brawny was severe enough to
reduce grain yields.
<D -I O to ^CO _ ® 7 CO C O m
39
Frold
\//A Brawny
c - control
Figure
10.
G r a i n yi e l d s
B H pi
I - infested
(* = s i g n i f i c a n t at
.05 level)
40
CONCLUSION
Infested Froid and Brawny winter wheat had higher
osmotic potentials and lower fructan contents than the
respective controls.
Therefore, one may infer that cold-,
hardiness was reduced by a £>. noxia infestation. The
inference for infested Brawny was corroborated by the field
survival and grain yield trials which indicated survival and
yields were reduced by an infestation.
An. infestation did
not significantly affect survival or yields for Froid or PI
372129.
The only significant effect of a D. noxia
infestation on PI 372129 was a reduced sucrose content. Thus
it appeared that PI 372129 was not completely resistant to
affects of D. noxia but is resistant to affects on fructan
accumulation, osmotic potential, field survival and grain
yield.
This implies that the resistant trait of PI 372129
confered resistance to a decreased level of cold-hardiness
caused by an infestation.
Extrapolations from this investigation are relatively
general due to variables which may alter the conclusions
discussed in this thesis.
D. noxia population levels and
winter-freeze conditions are variables in particular that
may drastically change at different locations and times.
From this investigation, one may surmise that D. noxia has
the capability to reduce the cold-hardiness of winter wheat,
41
though according to field survival and grain yield results
of Froid, this reduction does not necessarily translate
into significant survival and yield reductions.
If hardier
varieties like Froid are able to tolerate a certain level of
cold-hardiness reduction caused by an autumn infestation,
these varieties may be considered an option to mitigate
winterkill.
Finally,
it appears that PI 372129 offers a
source of germplasm that is resistant to effects of D. noxia
on cold-hardiness, so its use in current backcross breeding
programs may provide a prefered option when resistant
varieties become available.
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43
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44.
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49.
Thomas', K.J. and G.A. Taylor.
Unpublished data.
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49
APPENDIX
50
Table 3.
Temperature and Precipitation data for
1990/1991 growing season in Bozeman, Montana37.
temperature 0C
date
precipitation
X maximum
X minimum
X
Aug. 1990
82.30
51.77
66.95
1.34
Sept.
78.70
47.50
63.10
0.32
Oct.
60.20
34.00
47.10
1.38
Nov.
48.40
27.50
, 38.00
1.63
Dec.
26.80
3.90
15.40
0.91
Jan. 1991
32.90
14.00
23.50
0.64
Feb.
48.00
27.70
37.85
0.52
Mar.
45.20
24.00
34.66
1.83
Ap.
52.06
31.06
41.56
3.04
May
61.25
38.93
50.09
5.25
June
71.96
46.03
59.00
1.88
July
84.90
52.90
68.90
0.50
Aug.
86.96
54.30
70.60
0.46
.
X
I
51
Table 4.
Mean squares for osmotic potential measurement
(in bars) after a simulated hardening period.
source
df
mean souare
D-value
block
3
1.74
treatment
I
101.31
whole plot error
3
0.96
variety
2
90.64
.0000
blk*var
6
1.30
.7700
trt*var
2
18.32
.0008
blk*trt*var
6
0.53
.9662
70
2.33
subplot error
.0000
52
Table 5.
Mean squares for osmotic potential measurement
(in bars) after a simulated freeze period.
source
df
mean scruare
D-value
block
3
6.64
treatment
I
51.94
whole plot error
3
9.46
variety
2
92.39
.0000
blk*var
6
3.35
.0907
trt*var
2
33.92
.0000
blk*trt*var
6
2.77
.1655
71
1.75
subplot error
.0000
53
Table 6.
Mean squares for fructan content (in mg/100 mg)
after a hardening period in the field.
source
df
mean souare
n-value
block
3
1.40
treatment
I
47.07
whole plot error
3
1.90
variety
2
138.30
.0000
trt*var
2
16.48
.0121
12
2.52
subplot error
.0155
54
Table 7.
Mean squares for sucrose content (in mg/lOOmg)
after a hardening period in the field.
source_______________ df
mean square______p-value
block
3
2.10
treatment
I
6.80
whole plot error
3
0.08
variety
2
1.00
.0222
trt*var
2
0.54
.0944
12
0.19
subplot error
.0030
Table 8.
Mean squares for fructan content (in mg/lOOmg)
after a freeze period in the field.
source
df
mean sauare
n -value
block
3
2.27
treatment
I
26.08
whole plot error
3
3.47
variety
2
330.34
.0000
trt*var
2
16.50
.0715
12
4.98
subplot error
.0712
56
Table 9.
Mean squares for sucrose content (mg/lOOmg) after
a freeze in the field.
source
df
mean scruare
D-value
block
3
O'. 3 0
treatment
I
5.10
whole plot error
3
0.04
variety
2
1.07
.0138
trt*var
2
0.76
.0361
12
0.17
subplot error
.0016
57
Table 10.
Mean squares for field survival (in percentage)
after a winter season.
source
df
block
3
779.28
.0333
treatment
I
22226.00
.0000
whole plot error
3
588.45
variety
2
18973.00
.0000
blk*var
6
2702.70
.0000
trt*var
2
3936.30
.0000
120
259.60
subplot error
mean scruare
n-value
58
Table 11.
Mean squares for grain yield (in bushels/acre)
after a summer growing season.
source
df
mean sauare
o-value
block
3
216.70
treatment
I
492.86
whole plot error
3
100.90
variety
2
525.20
.0152
trt*var
2
116.00
.2990
12
86.73
subplot error
.1141
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