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5/08 K/Proj/AocaxPres/CIRC/Dateouemw

ABSTRACT
SEASONAL RESPONSES )F SOLUBLE CARBOHYDRATES IN THE LEAVES OF
FOUR COOLuSBASCN CRiSSES TO FIVE NITROGEN TREATHENTS

by David S. Green

The quantitative effects of applying 0, 3, 6, 9 and 12 pounds of
actual nitrogen per thousand square feet on the soluble sugars in the
leaves of Agrostis palu: ris, Festuca rubra, Loliungperenne and £23
pratens's were studied to determine the degree of carbohydrate depletion
at higher nitrogen treatments. The mono-, di-, and oliogosaccharides
were extracted with boiling 80 percent ethanol, separated via paper
chromatograms, and the resulting sugar Spots evaluated quantitatively
with a densitometer. The polysaccharide fraction was observed to be a
glucopolvfructan, extracted with boiling water and quantified colorinet-
ricallv by a ketohexose test.
The greenhouse environment produced leaf tissue considerably lower
in oligosaccharide than field samples. Effects attributable to nitrogen
treatments were most prominent in the oligosaccharide fraction, particuc
larly oligosaccharides other than sucrose. The di- and monosaccharides
failed to produce concentration differentials directly attributable to the
nitrogen treatments. Treatments providing more than one and one-half
pounds of actual nitrogen per thousand square feet per application pro-
duced near identical carbohydrate reaponses. Regrowth in the dark estimates
of food reserve agreed favourably with chemical determinations, particu—
larly values for oligosaccharide minus the sucrose fraction. Temperatures

in the 80 F range and higher appeared to exert an adverse effect on prowth

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SE SUNAL RESPONSES OF SOLUBLE CARBOHYDRATES IN THE LEAVES OF

FUUR COOL-SLASON GRASSES TO FIVE NITROGEN TREATMENTS

By

DaVid Go Green

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Crop Science

1963

QM“; fl ’0 0
Approved: " 47 v . [Lilla 2/

I

I

ACKN 0W LE GE KENT

The author expresses appreciation to Drs. J. B. Beard, C. R. Olien,

C. J. Pollard and H. M. Brown for advice and encouragement during the

course of this study.
A special consideration is given to my wife, Dixie, for assistance

in conducting the statistical analysis, and in preparing the manuscript.

Ho
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INT R0 DU CT I 0:!

One of the objectives of nitrogen fertilization of grasses is to
maintain plant growth. This growth is a result of the interaction between
nitrogen taken up from the soil and reserve food materials already avail-
able to the plant. The utilization of nutrients, primarily nitrogen,
absorbed from the soil solution requires metabolic energy and a supply
of carbon skeletons, Specifically organic acids. The major storage forms
of this energy and organic acids are the soluble sugars, which are re-
ferred to as the carbohydrate reserve, or more generally, reserve food
material. The objectives of this study were; 1) to determine the nit-
rogen treatments at which the carbohydrate reserve in the leaves became
a grouth~limiting factor, and 2) to determine the quantitative effects
of various nitrOgen treatments on these individual leaf sugars under

greenhouse and field environments.

REVIEN 0? THE LITERATURE

The importance of the sugars and sugar polymers, which are referred
to as reserve or storage carbohydrates, to the utilization of available
nitrogen by the plant is most easily elucidated by consulting a general
textbook of bioctemistry. Carbohydrates can be broken down via the
glycolytic (Enbden, Hyertmfi; Parnas) pathway and the citric acid (Krebs)
cycle to various organic acids such as pyruvic, (X.-ketoglutaric,
fumaric and oxaloacetic acids. These organic acids may combine by well

Y“:

established enzymatic reactions with ammonia, AT: and in the presence of
magnesium ions form the corresponding amino acids. Chlorophyll synthesis
from glycine and succinic acid is another reaction which uses organic
acids. ATP, which stores the energy for these synthetic reactions, is
also a product of this carbohydrate catabolism. Without either the carbo-
hydrate to supply the energy and organic acids or a supply of available
nitrOgen, the plant is in an unfavorable position to carry out the syn»
thesis of new materials.

Thomas (cited by Troughton, 23) defizes a reserve food material as a
substance that has a preliminary period of accumulation followed by a
period in which the substance is maintained in_§i£2_at a relatively high
concentration, and that later, in association with physiological processes
taking place in the immediate vicinity or elsewhere, the concentration
diminishes. According to Weinman (cited by Troughton, 23) certain groups
of carbohydrates viz. sugars, fructosans, dextrins and starch are the

most important reserve substances in grasses. Pentosans, hemicellusose

and true cellulose were considered structural materials and not further
utilizable as food reserve by the plant. McCarty, Brown, (both cited by
Troughton, 23) and Sullivan and Sprague (22) hold similar opinions. The
dominant form of reserve carbohydrate in cool season grasses according to
De Cugnac's 1931 (a) classification are fructosans. Chemically the type
of fructosan in grasses for the most part is the phlein structure, that
is with a carbon 2, 6 linkage as contrasted to inulin's carbon 1 to
carbon 2 linkage (9).

Archhold's (l) exhaustive review of fructosans in monocotyledons cov-
ers research prior to 1953. Troughton's (23) 1957 review of the under-
ground organs of herbage grasses cites three workers, McCarty and Weinman,
Mollvanie, and Aldous, who studied seasonal affects on food reserve
material. They noted a decrease in concentration of reserve carbohydrates
in the roots concurrent with early shoot growth and then a gradual in—
crease during the late spring and summer followed by a decrease at the
time of secondary herbage growth. Mollvanie associated maximum reducing
sugars with rapid vegetative growth, maximum sucrose with differentiation
and greatest quantities of "reserve polysaccharide" with the brief rest per-
iod prior to secondary growth.

These three authors plus Bews and Bayer, (23) and Arber (23) all
noted carbohydrates are accumulated in the roots during the autumn.

Sprague and Sullivan (21) noted in orchard grass that high-nitrogen
fertilisation tended to reduce the percentage of fructan and low-nitrOgen
fertilization tended to increase it. They also noted that the stubble

and lower 2/3 of leaf blades contained the highest weights of fructan.

The roots and upper 1/3 of the leaves were much lower in fructan. Sucrose
was highest in the upper 1/3 of the leaves, and next highest in the roots.
An inverse sucrose-fructan relationship existed. Sullivan and Sprague
(22) also noted the regularity of loss of fructosan with temperature in-
crease was similar in both stubble and roots.

Baker (23) working with Lolium perenne also observed that the per-

 

centage of total soluble carbohydrates was always greater in the stubble
than in leaves or roots. Shara and Tanaka (6) noted the fructosan con-
tents of each organ of Italian ryegrass, Timothy, Bermuda grass and Bahia
grass decreased as temperature rose, the concentration of fructosan in

all four grasses being higher in stubble than in other plant organs.
Hansen, 3£;_§l;, (9) refer to Waite and Boyd's observation that the stems
of growing plants commonly contain much higher levels of fructose polymers
than do the leaves.

A direct method to measure organic food reserves in relation to growth
of alfalfa was develOped by Graber, 33:.Ei;9 (8). They transplanted
alfalfa plants into pots which were placed in a dark growth room. Before
and after dark regrowth values were recorded for dry matter, total sugar,
dextrins and soluble carbohydrates, and total nitrogen. They concluded
no very specific conclusions could be made from these studies £33153) but
that these procedures might be useful when interpreting results from chem-
ical determinations for plant food reserves. Harrison, (10) and Juska
(12) have used this method for evaluating bluegrass food reserves.

Jordan (ll),used a modified version of the McPary and Slattery (18)

Seliwanoff test for ketoses to study the effect of environmental factors

on the carbohydrate reserves in the leaves of bentgrass (Agrostis
2alust~is). He concluded fructan (phlein type fructosan) to be the

best indicator of reserve carbohydrate in bentgrass, total fructose next
in order of reliability, with free fructose and fructose from sucrose

of least importance. A constant temperature of 70 degrees Farhenheit
produced the optimal growth response with the corresponding negative
effect on reserve carbohydrates.

The discovery of paper chromatonraphy by Martin and Synge (15) and
its subsequent application to carbohydrates provided a technique whereby
the individual fractions of the carbohydrate reserve could be studied.
Using these techniques as adopted by Laidlaw and Reid (13), Lopatecki
SE; al;_(15) followed the quantitative changes of soluble carbohydrates
in the stems of three wheat varieties during two growing seasons. They
noted the presence of glucose, fructose, sucrose, oligosaccharide and
fructosan (phlein type). The oligosaccharides (a number of low molecu-
lar weight nmnoglucopolyfructans) soluble in 80 percent ethanol were the
dominant form of carbohydrate stored in the stems prior to ripening of
the grain. Glucose showed maximum accumulation around heading and pro-
gressively decreased towards maturity. Fructose showed two peaks of
accumulation, the first at heading and second during the dough stage.
Sucrose concentration was appreciable at heading and gradually increased
throughout the milk stage when monosaccharides were decreasing. Accumu-
lation of fructosan in stems occurred after the initial build-up of
sucrose. Oligosaccharide trends paralleled fructosan but in much greater

quantities.

These references illustrate that the relationships between nitrogen
availability and total soluble carbohydrate levels have long been estab-
lished. They also indicate a lack of information regarding the effect
of nitrogen on the carbohydrate fractions of which this soluble carbo-

hydrate or food reserve is composed.

SECTION I
THE INFLUENCE OF NITROGEN TREATMENTS ON THE SOLUBLE CARBOHYDRATES IN

LEAVES OF LOLIUM PERENNE, AGROSTIS PALUSTPIS AND POA PRATENSIS

 

 

UNDER GREENHOUSE CONDITIONS

The objects of this experiment were; 1) to develop a suitable tech-
nique for analyzing large numbers of samples for soluble carbohydrates,
and 2) to observe the quantitative effect of varying nitronen treatments

on these carbohydrates under greenhouse conditions

MATERIALS AND METHODS
Plant Culture Methods
Pure stands of bentgrass (Agrostis palustris, var. Toronto), bluegrass

(Poa pratensis, var. Eerion) and ryegrass (Lolium‘perenne9 var. common)

 

 

were planted October 15, 1962. The seeding rate used for the bluegrass

and ryegrass provided 2,500,000 yerminatable seeds per thousand square feet.
The bentgrass was vegetatively planted. A randomized block experimental
design was used with three replications. The flats were 8 inches deep by

9 inches by 2“ inches containing a soil mixture of 50 percent sand, #0
percent loam soil and 10 percent vermiculite by volune. Approximately u
pounds of 15-15-15 per thousand square feet (.6 pounds actual nitrozen,
phosphorous and potassium) was mixed into the tap 2 inches of soil in the
flat prior to planting. The grasses were cut weekly to 1 inch height of
cut with hand clippers. Water and fungicides (weekly application of Hildex
from December 15 to May 27) were applied as needed. The plants received

no artificial illumination during the course of the experiment.

No additional fertilizer was added to the treatment flats between Oct-
ober 15, 1962 and Harch 2S, 1963. On March 25 a complete harvest was taken
for chemical analysi . Treatments totalling 0, 3, 6, 9, and 12 pounds of
actual nitrogen per thousand square feet using ammonium nitrate dissolved
in two litres of water as the nitrogenous material were then applied in
one application. Subsequent harvests were taken April 6, April 13, May 1,
and Hay 27.

Analytical Methods

The selection of a suitable analytical technique was governed by the
number of samples to be analyzed and the degree of accuracy required for
following seasonal variations in soluble carbohydrates in a realistic
fasnion. “he analytical procedures employed by Lepatecki §£:-§fl:_(15)
were based largely upon methods previously reported by Laidlaw and Reid
(13). Laidlaw and Reid’s (1H) extraction and elution technique was com-
bined with Dubois EELuiiL (5) phenol-sulphuric acid colorimetric deter—
mination and consistently accurate results were obtained. A more expedient
method combined Laidlaw and Reid's extraction and separation on descending
paper chromatograms with the utilization of a direct photometric reading
as described by McFarren, Brand and Rutkowski (17), but without a record-
ing integrator. This method eliminated the time required to locate and
elute the sugars from the paper for colorimetric determination.

A procedure evaluation using D-xylose showed an average recovery for
eight replications of 87.32 percent with a variation of 8 percent. These
values were judged to be satisfactory for seasonal carbohydrate studies.

A comparison of extraction methods using a Soxhlet apparatus versxs

‘

extraction over a suction :iltrr 3rd Buohner vacuum flask produced results

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alcohol fraction contained mono-, di- and oligoeaccharidee. If
tissue moistures w thin one grass variety varied more than 1 per-
cent between treatments, calculated percent moisture and adfiusted
to a final 80 1e rcent ethanol concentration in the filtr te.
Water fraction. Extracted with 150 ml. boiling water the filter
reeidue from C-2. This water fm ion contained the polvsaccrlarides.
Rade alcohol fraction to 230 31., sampled 50 ml. aliquot and evap-
orated in Flash Ev Mp meter at reduced pressure and 65 deqreee C.
to 10 ml. Similarly made water fraction to 150 31., sampled 30
ml. aliquot and evep ratei to 10 ml.
Hydrolysis. Divived alcohol fraction into two 5 ml. volunes.
Nade one 5 ml. volune 1 normal with HCL. Similarly divided 10
31. W3ter fraction int 2 equal volu3cs and hydrolyzed one fraction
as ahove.

ration
Prefiared No. l Whatran (18-1/4 inch by 22-1/2 inch) as follows;

drew lines at 1 inch, 2-1/2 inches, and a inches from tap of paper.

Labelled paper and spots (kept 1 inch min inm separation between

Folded paper and placed hair drier (cold air) on Opposite ends of
origin line. Us ng l :1mb d3 pipette 3 Spotted 50 lambda volumes of
alcohol and water fractions from C-S to labelled origin. Similarly
spotted 3tan {ards (19, SO. and 100 micrograms glucose , fructose,

1

sucrose, and D‘xvloee).

Clipped paper to antisiphon rod and lowered into dry solvent

trough in Chronatocab. Placed sapport bar in trough. Adde ed solvent

10

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6)

7)

Percent Dry %.'

(7) to trough and removed antisiphon rod clips (solvent used;
n-propanol, benzyl alcohol, 85 percent ormic acid and water
50/72/20/20 v/v). DevelOPed paper for 30 hours.

Removed paper by fastening clips to paper and antisiphon rod and

placed in drying hood (room temperature) for one hour.

{1)

prayed with p-anisidine indicator (1 gram p—anisidlne in 40 ml.
n-butanol, plus 2 ml. 19 percent HCL). Dried 1 hour in hood.
Color was developed by placing paper 3-5 minutes in oven at
95-100 degrees C.

Rcanned spots under Photovolt Densitometer (model 52~d) using a
”45 mm. filter, and recoriea maximum optical density. Con-
structcd graph with Optical density as the ordinate and concen-
tretion as the abscissa using readinqs from the standard spots.
Converted optical density readings of unknowns directly to sugar

weights per 50 microlitrea.

 

 

m€rrozrams sugar x 100 = micrOgrame sugar 2 percent dry
10,000 tines percent moisture moisture weight

11

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Soluble carbohydrate percent of dry weights for Toronto creeping
bentgrass are in Table 2. Glucose and fructose levels were similar re-
gardless of nitrogen availability. However, between Ray 1 and 27 Fructose
in the O nitrOgen treatment showed a significant increase. Fructosan
treatments showed an initially Wide divergence on April 6, but by April 13
the 3, 6, 9, and 12 pound treatnents had formed one group while the O
treatnent maintained a greater value. fay 27 the 3 pound treatment also
increased in value. The increase in fructose corresponds to the increase
in fructosan, the fructose increase showing a lag in response relative to
higher fructosan levels.

Sucrose, the dominant carbohydrate did not show a consistent response
to the nitrogen treatments. As with the Marion kcntucky bluegrass, sucrose
trends in the bentgrass paralleled the check treatment.

Total carbohydrate with the exception of the April 6 harvest was high-
est in the G nitrogen treatment throughoat the course of the experiment.

Traces of oligosaccharide were noted in all bentgrass plots prior to
fertilization March 25. Ray 1 the 0 treatment and May 27 the O and 3 pound

treatnents contained oligosaccharide. Environmental factors in ddition

m

to applied nitrogen eftected the oligosaccharide fraction. May 27 the 3

pound treatment again contained oligosaccharide, presumably an indication

of depletion of available nitrogen. The disappearance of oligosaccharide

in the 0 treatment on April 6 and 13 is attributed to environmental factors.
Table 3 represents soluble carbohydrates for common perennial ryegrass,

under conditions identical to the Marion kentucky bluegrass and Toronto

creeping bentgrass. No oligosaccharide was present in leaf extracts.

Fructose and fructosan values were very low in all nitrogen treatments.

14

Table 2. Leaf carbohvurate nercantarafi deterfiinefl an n drv weicht
hasia occurring in Toronto creeping beuttrags unier five ni€rnren treat»
manta. IQBS.

?oundfi fiitrovnn

 

 

 

 

 

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flarcr‘ I.‘ 1981. .70 0'0 10”“ ti. .0

25
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nshla 3. Lea? cerhchvfirata parcantares fiaterW1nQd on a dry weight
occurring in common perennial rvahrazs unier five nirvancn treat-
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The 0 treatment recorded slighly higher fructose and fructosan levals.
Glucose values, while greater than fructose and fructosan did not illus-
trate a varying response to nitrogen. Sucrose was the dominant carbo-
hydrate in ryegrass under grennhouse coaditions. However, a poor

correlation existed between soil nitragcn levels and carbohydrate values.

17

“Ian-v; :r-f’r 33¢

*al carbohydrate valuaa for all nitrocen truatnants peanrally were
lo'e* tian the cane .k treat!¢nt in all three :r33389. ho confl’ tent d1”-
fercnces bgtueen nitrogen tPGQtT ant; occuzre d (exclu$2nz chagk tcwatwent).
”xxtv-t.fies days Pfter nl trc,:. a plication the 3 sound treatwent values
for fetal Carbohyflrata weie interwefiiata betx¢en the 6, 9, ahi 12 group-
ing an! 0 treatment for Tfironto cremginn bentwrama (T4315 2). Hitwcgan
'\pIetian in the 3 pound treat/tut is aviiencad lalirectly Lv thifi accuvu-

_I

latic a CF car no‘;3rate. W1 :9 3.1513 nitrcxen 3:;licativn .33 d,,-3rk to prev

vice «news; nitrtgca fcr plant uptake in all trqat eats aufi hat can

anPSfan trnetmcnt (.xclu fim my the Check), diFCUrunceg fllL no: Cflfid".
Sucrasa was the dominant sugar in the thrua firnfilfifi (T films 1, 2,
ani 3). Tchtwssn Far Varzun kentuckv bluvfrun; and Twrfinto cree5115 rant-

...~..',. \- .-'. .,.. . . ’ . ',’. , ‘ ._ ‘V'fl ..., .. q 1 ... '. '
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Tuvy o sarved tucraae :0 be aw3r9x7mntaly QUU71 ta Fructu _nwn in the uyrar
two«thirdn of lea? blxdae, hut far all lower leaf and 3tunble 'srfq
Fructomm wait: -."‘!o:-3r;2-3;'-t over r'xcr-c 59 «1:11 t‘e other 1m 3:3 r-t‘enent. Can-
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Hanna” 0?. 31. (3) T0 it cut tfimt marrow: in the "0 t 1 .P““t" , Iatfi in
Quantlt“ and In impurtance, to filsrt fifltxzfilinm. There is evi Race that
fiucrqu 13 utilizfid in the Forwaticn of Fructwnn by trflqs“ructezvlnt§mw.
3:1 lemi’ ti.;~:s::.;-a, :;- . rm 3 value":

T‘mue cont: 302'21‘103’5 era ‘M’l‘. “4 way m as

{#:3231th 1:31;!) Fructfivusn were? LllrtTafiiiifz‘A

...:
DJ

Jordan's studies based on leaf tissue of Agoetisgpaluetriq indicated

 

fructan to he a much better indicator of reserve carbohyflrate in bent—
grass than sucrose. Peach and Tracey (20) pointed out that the FcVary
and Slattery method for analysis of fructonans can be used only when 80
percent ethanol insoluble fructosans are present. Jordan used the shove
method on material extracted with water (90 degrees C. for 30 to 60 min-
utes). This extract would contain 80 percent ethanol soluble sugars. The
possibility exists than Jordan's values for sucrose are low.
Oligosaocharides were noted in the Toronto creeping benterase on
March 75, May 1 and May 27, and also in Marion bluegrass in preliminary
investigations, but not between March 25 and Way 27. No oligosaccharide
and only low values were recorded for fructosan in ryegrass. Bacon (2)
also reported the presence of fructosans in Lolinm‘perenne but little
oligosacoharile. He also noted raffinose to be present in green rverrass
leaves. Paf€inone was not observed in any grass during greenhouse studies.
The low values for oligosaccharide are attributei to environmental factors

other than nitrogen treatrents.

19

SECTION II
THE IfiFLUENCE OF NITFM .W TREATHEKTS ON THE SOLUBLE CAR THEDRITE fl4nCTIONS

IN LZAVES OF A.3 P0 ”IS PARLSTPIS, FESTZCA PUBRA VD DOA Poiteqstg

 

 

UNDER FIELD CO-NDITIQ

The olmjc ives oft is experinental series were; 1) to improve the

ana ‘.ytical methods used in section 1 for determining fructosan, 2) to
observe the quantitative eff eat of varyinr nitrogen treatments on the
solitle carbohydrates under environmental conditions, 3) to compare the
addition of soluble nitrogen to turf in single seasonal app olic ations
versus fractional amounts di:t Mi)ited over a five-month period, and u)
to comaare the technique of measuring plant food reserve as inlicated by
regrowth in the d31k with carbohydrate reserve values determined by the

chemical methods later described.

fiATERIALS AND METHODS
Plant Culture Methods
These eXperinents were located at the Crop Science Farm, I'ir chigan
State Univer Sltf. Prior to 3961 the plot area was a low management eras
award. This turf was removed with a see cutter and the soil augmentei with
coarse sand to produce a sand 7 loam soil to a six to eight inch depth. The
area has 3 1/900 foot slooe wifi c1: proviies adeQJa esurfaco drainafe.
Toronto creeping bentgrass was dormant planteo November 1951. Penn-
1awn creepinr red fescue, Nerion kentuoky bluegrass and common kentucky
bluegrass were seeded at 2,500,000 8 code per thousand square feet (seed and

stolen source, Hiram F. Coiwin and Sons, Detroit). The e'perimental design

was a randomized block with three replications. Individual plots measured
three foot by 2% feet. These areas received four pounds actual nitrogen

prior to August 18, 1982.

su-

D ffsrontial nitrogen treatments of O, 3, 6, 9, and 12 pounds per
thousand square feet per season of available nitrogen (ammonium nitrate
carrier) were applied with a Scott's 3 foot spreader on August 18 and
September in, 1962, (each application 50 percent of seasonal total).

The 1083 treatments were applied in one-sixth season's total nitrogen amounts
April 18, Kay 15 and 31, June 15, July 16 and August 15. The ammonium ni-
trate was watered immediately after arplication to prevent foliar burn to

the leaves. Analytical sanmlcs were harvested June 8 and 22, July 8 and 23,
August 8 and 22, 1963. In addition, the Marion kentucky bluegrass was
sampled Has 11 and 23.

Similar methods were used to apply 0, 6, and 12 pounds per thousand
Square feet of actual nitrogen in single treatments August 5, 1963.

No foliar burn occurod. Samples were obtained August 5, 6, 8, 10, 12 and 16
and analyzed by the methods subsequently described.

The bluegrass and fescue areas were mowed at one and one—half inches
twice weekly. The bentgrass was mowed four times per week at one quarter
inch. Supplemental moisture was supolied by sprinkler irrigation. Other
maintenance practices such as herbicide and fungicide treatments were ap-
olied as required.

Soil temperature data was provided by the Department of Agricultural

,ering's microclimate station located at the Horticulture Farm,

2'1
:5
“’3
~40
:3

Michigan State University.

21

Analytical hethods

A modified version of the analytical technioues descrit ed in Section I

1-:-

11‘:

was used for all field samples. Experiments were conducted to determ n

the sugar moieties wnich comp03ed the water fraction (polysaccnar ide) i

:5

the three grasses. Water fractions of Toronto creeping bent; r-3. and

Marion kentucky bluegrass (Section I, analytical nethois can) we} e applied
in a 15 inch solid orig in line to Whatman No. l chromatographic filter
paper as described in Section I, analytical methods D. The paper was run
for 133 hours using descending n—proganol, benzyl alcohol, 85 percent Fortic

acid, and water (50/72/20/20 v/v) as the developing: solvent. Parker st pipe

4

1 inch by 22-1/2 inches were developed with p-m 111s id.ine-HCL to locate
non-migratory origin lines. The origins were eluted usi.ng th: tet Minion

of L? idlaw and Reid (1n),hydr01yzei with l N HCL and tech romatOgraphed.
Cltcose and fructose standazds we1 e alr so included. The only super remain»

1mg after this processing wan $ructose. Clucose was not observed. Fructose
was concluded to be the dominant sugar moiety in the water fra 3t.on. 1he
McR.3rV and Slattery (12 ) WJth:Dd for the colori: wetric determination of
fructosan was used to evaluate the water frgaction. This modiric xti1on elim-
neted the photometric determinatic n of the hydrolrzed wet or fraction.
Determination

A. Samples

1) Sampled 10 grams frc h material at 10 a.m. on clear d¢»a s.

z:
.1)
in
'F
t f

Harvested Fescue and blw 3820 s with a mower set to 1-1/2 inch

height of cut; oentgrnas was cut at 1/3 inch.

2) Denatured ”or 5 m1nuto: in boiling 80 percent ethanol.

B.

C.

D.

3) Stoppered and stored in refrigerator (minus 9 degrees F.).

Extraction

l) A4193 2 microgram per 1 microlitre of D-xvlose to A-3 as a
recovery check.

2) Hacerated Aa3 for 3 minutes in a Waring Blender.

3) Alcohol Fraction. Poured B-l through Buckner funnel (1 sheet
Nhatman No. I filter paper) connected to a vacuum flask. When

dry aided 150 ml. boilin;

."
...
..J

80 percent ethanol. This fraction
contained the mono—, di- and oligosaccharides.

N) Water fraction. Extracted (Buchner funnel, a second vacuum flask)
resifiue from 8—3 with 150 VI. boiling water. This fraction con-
tained the polysaccharides.

S) The alcohol fraction was made to 250 ml., 100 ml. alicuot sampled
and reduced unuer vacuum in a Flash Evaporator to 10 m1.

6) Hydrolysis. he 10 ml. alcohol fraction was equally civided.

7) One 5 ml. sample was made 1 N with “CL and left overnight at room
temperature. Both the hydrolyzed and unhydrolyzed samples were
spotted as in C.

eparation

Identical to Section I, analytical mathofls D, only 25 microlitnes

Spotted (versus 50 microlltpas in Section I).

Percent Dry weight.

Iientical to Section I, analytical methods E.

Water Fraction

3) .25, .5, l, 2, 3, a, and 5 ml. of water fraction E-u were

pipetted into 30 ml. teat tubes and made to 5 ml. Prepared a

watar blank.

y-la

‘3 ‘3.

IV

5.2..

551 5 wl. G. yarcent alcoho c reaorcinnl (1 Vrac PEHUFCEWOI

car 1 litvv 33 oarsaat at anrd).

3) tidied 1": ml. 3-" fror‘wgaxw 'fi. (.1 Vultma 1'? 1.6:: water) to 5 vol-
ufiefi of concoztaitafi hob). Jinn! Taur‘tiweq witw stiftiné r05.

u) Placed in E) 3yfirfiflfi C. water bath for l7 rinutez.

ratical fianrity at Sun mu. (hauach

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in a dark growth chamber at 70 degrees Fahrenheit, June 15, 1963. The
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ly. On July 11, after 26 davs in the dark, the height in inches of re-
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was subsequently removed, dried, and weighed.

25

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were near 1v 1d mtical. This would indicate that sucrose almost eyclueivew
1y made up the olinosacch aride fraction. Also, cligosaccharide values For
June 8th to August “in :‘eviated vzry 3.ittle from the 2 percent level.
Fructosan was present only in trace an arts. Total carbohyflrate leuels
decreased ve ry sharplv from Jute 8 unti.l July 2 . This decline paralleled
the depletion in the monos cc Wari fractions

Figures 3-A to 3-H illustrate dry weights for the solutle carbohy-
drates in Toronto creeping ten1:cr% s. Glucose, fructose, and sucrose show
definite seasonal var mtion ,but lack differences attributable to the
nitrogen applied. Only trace amounts of fructosan were preeent. Progres‘
sively higher oligoeaccharide values resulted with decreasing nitrogen
treatments. Total carboh; drate values shoaed two peaks of accumulation;
the first in late June and the second in late August.

Sucrose, glucose and fructose concentrations in Pennlawn creeping red
fescue (Figures u-A, u~c and nut) were independent of the nitrogen treat-
ments. Sucrose values maintained approximately 1.5 perce ant dry we i; ht
throughout the s 1mmer season. Glucose and fructose decreased from a June 8
peak to trace values in rid-July. As light recover} in fructose w: 15 noted
in late Atzgust. Oligosaccharide (Figlre u-D) values shocefi a tenlency to

P

maintain values near 2 perce at, but lac}:ed a pattern related to the nitro-
gen treatments. Total carbohyflrate values produced the most strik ir .r re-
su .ts. On May 23 the 0 treatment was o.u to 0.8 perCc ant ill Wher than the 3,
5, 9 and 12 treatments. On June 8 the 6, 9 and 12 treatments recorded a

peakv value (1 percent greater than the 0 and 3 treatments), and then do-

clined until August 3. The O and 3 treatments showed a lag effect in

29

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reaching their peak concentration on June 22, after which they similarly de-
clined. The 3 treatment was intermediate between the O and 6, 9 and 1?
treatments June 8 and 22.

The results of applying 0, 6 and 12 pounds of actual nitrogen per
thousand square feet per season in one application August 5, 1963, are i1-
lustrated (Appendix Table VII for Marion kentucky bluegrass. In general,
the 0 treatment values for all four carbohydrates were higher than the 6
and 12 treatments. The 12 treatment depressed the concentration of sucrose
oligosaccharide and fructosan more rapidly than the 6. Seven days after
nitrOgen application, vegetative growth and carbohydrate resoonses were
indistinguishable for the 6 and 12 reatments.

Common kentucky bluegrass (Appendix Table VIII) maintained relatively
stable values for oligosaccharide (predominantly sucrose), irrespective of
the nitrogen treatment. Fructosan values were less than o.u percent. Less
growth response in terms of green colour and vegetative growth was observed
for the common as contrasted to the Marion kentucky bluegrass. Merion
contained both sucrose and longer chain oligosaccharides. Common kentucky
bluegrass contained only the sucrose oligosaccharide August 5 to lo.

Sucrose concentrations for Toronto creeping bentgrass (Appendix Table
IX) did not vary more than 0.4 percent between the three UltPOgen treat-
ments. The 0 treatment oligosaccharide concentration contained 1 percent
more sugar three days after nitrogen application than did the 6 and 12
treatments. This large differential continued for the next nine days (ex—
periment then terminated). The highest values observed for fructoee and

fructosan were 0.15 percent and 0.09 percent respectively.

32

Sucrose (Appendivaahle X) was the predOminant oligosaccharlde in
Pennlawn creeping red fescue August 5 to 16. No differential responses
attributable to nitrogen treatments were observed. Glucose concentrations
ranged from 0.2 percent to 1.5 percent with an over—ell increase in values
from August 5 to August 16. during this period, soil temperatures at the
3 inch depth decreased from 90 to 70 degrees Fahrenheit. No consistent ni-
trogen response was observed.

Results of the regrowth in the dark experiment (Appendix Table V) for

the bluegrasses and bentgrass showed greater height and weight of regrowth

occured in the lower nitrOgen treatments.

DISCUSSION

Glucose, fructose and sucrose are not typical reserve food materials
according to Thomas' definition. These sugars are active metabolically
as intermediates between the initial products of photosynthesis and
storage forms of carbohydrates, specifically oligo- and polysaccharides.
Values for glucose, fructose and sucrose (Figures 1, 2, 3 and u) for all
four grasses studied under environmental conditions failed to exhibit
characteristics directly attributable to the nitrogen treatments. Con~
versely, oligosaccharide, and fructosan when present, were in highest

oncentrations in treatments with the least nitrogen applied.

Leaf tissue, as Sprague and Sullivan (21), Baker (23), Shara and
Tanakea (B), Hansen 33;.El;.(9)9 and others have noted, is lower in
fructose polymers than is the stubble. This may account for the low
oligosaccharide and fructosan values encountered. Leaves of Toronto
creeping bentgrass and Marion kentucky bluegrass contained more sugar
polymers than common kentueky bluegrass and Pennlawn creeping red fescue,
and also produced more consistent carbohydrate responses attributable to
nitrogen treatments. Marion kentucky bluegrass and Toronto creeping bent:
grass responded faster in terms of vegetative growth to nitrogen than dim
common kentucky bluegrass and Pennlawn creeping red fescue. This possi-
bly was due to the availability of leaf oligosaccharide. Carbohydrate
differences between treatments during May for Herion and Pennlawn were
negligible. Soil leaching and plant growth during autumn of 1352 nulli-

fied nitrozen differentials established in August and September. April 15,

34

May 15 and May 31 treatments were necessary to restore these differentials.
After June 8 oligosaccharide trends in Merion bluegrass and Toronto bent—
grass were consistent, the low nitrogen treatments containing the highest
concentrations of oligosaccharide.

The single nitrogen applications of 0, 6 and 12 pound treatments
August 5, 1963, did not show any relationships between nitrogen treatments
for grasses lacking oligosaccharide other than sucrose and fructosan. The
12 treatment oligosaccharido concentrations of bentgrass and Marion ken-
tucky bluegrass, plus fructosan in the bluegrass drOpped below the O treat-
ment less than 2n hours after nitrogen application. The 6 treatment re-
quired more than us hours to produce similar results. No differences
could be noted between the 6 and 12 treatments during the balance of the
eXperiment. The tendency for the 6 and 12 treatments to parallel one
another also occured with 3, 6, 9 and 12 treatments applied in one appli—
cation as described in Section I. Nitrogen differentials for an experi-
ment conducted over a period of several months should be applied in several
applications to prevent the apparent excesses of nitrogen as observed in
Section I and during the single-application field experiment. The grasses
studied under the glasshouse environment lacked appreciable oligosaccharide
in their leaves, hence no rapid carbohydrate-nitrogen interactions similar
to those obtained in Marion and Toronto creeping bentgrass could be ex-
pected.

Jordan's study of Agrostisgpalustris under varying environmental
conditions did not record the presence of oligosaccharides, other than
sucrose. He concluded that fructan, the specific fructose polymer found

in monocotyledons, was the best carbohydrate indicator in bentgrass leaf

tissue. This was not the case under the environmental conditions en-
countered in this study. Oligosaccharide other than sucrose (Figure 5)
was the most reliable indicator of differential carbohydrate reserves.
Fructosan levels in leaf tissue were consistently low. Jordan's fail-
ure to note the oligosaccharide fraction is likely due to the inability
of the nethod he employed to clearly distinguish the several sugars pres-
ent in the alcohol extract.

Soil temperatures (Figure 5) at the three inch depth increased from
61 degrees F. hay 22 to 83 degrees F. June 8. Sucrose values For Marion
dropped from a mean of 3.u8 percent on May 22 to 0.56 percent on June 8.
The high nitrogen treatments showed the largest decrease, the O and 3
treatment combination drooping 1.86 percent versus the 9 and 12 combined

decrease of 2.53 percent. Soil temperatures (Figure 6) remained in the

U.)

high seventies and low eightie until August 12. Glucose and fructose

.
values for Marion and Pennlawn, (Figures 1 and u) in contrast to sucrose,
increased in concentration with increased soil temperatures. After June 8
(June 22 for bentgrass) glucose and fructose in the bluegrasses and fescue
decreased steadily until mid-August. Oligosaccharide regained values in
late June and August which were considerably above the June 8 depression,
and maintained these near constant levels regardless of the eightv degree
soil temperatures. The advent of warmer soil temperatures (Figure 6) in

a,

early June produced responses in oligosaccharide similar to those caused

P'

by nitrogen fertilization. ”his initial heat effect appears to have
diminished by late June and July as evidenced by a decrease in hexoses

and increases in values for sugar polymers. Higher temperatures appeared

 

 

 

 

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to produce an adverse e ‘Eect on perce . total carm ohvdrate val.ues For
he ion blue gras s and Fennlawn Fescue, with values decreasing from rid-
June until mid-August.

Kentucky bluegrass and ore -ep.1.ng red fw cue are cool season grasses.
Marion kentucky bluegrass and Toronto creeping bentgrass, while also cool
season grasses, generally Show less te endency to become dormant in hot
weather. Lopatecki and Mollvanie both noted tr e highest hexose values
to be associated with vegetative growth. Lower hexose values were noted
during maturation, differentiation and dormancy. Late May and early June
results with Marion and Pennlawn produced levels of hexoses which aqreed
with these worker's observations. All four grasses produced less vegeta-
tive material when 3 inch dept h 8011 temperatures reached the high seven-
ties. Herion and Toronto glucose and fructose values.J while .ow, were

igher than similar hexose values for canton kentucky bluerress and creco-
ing red fescue during; .igh temperature periods.

The total carbohydrate reserve generallv includes hexoses, Oligo-
saccharides, and polysaccharides. Earlier in this discussion the inclu~
sion of hexoses in the reserve ca_bohydrate cl ssification was questioned
due to their active role rat":ojl--‘ll". However, line to their tendency

to be most nrOminent in concentration durinp -eriods of maximum vegeta~
1' r~ ,

lation-

w

tive growth and of lowest concentration during rest periods, a
ship which suvgests a very direct a3so Hi tion with sugar polymers, they
represent an integral component of the total carbohydrate reserve.

Total carbohydrate reserve values recorded minimxxs with the l? treat-

ment in Marion kentucky bluegrass and Toronto creepirzg bentgrass of 1.u3

percent and 1.02 pence ent respectively. Nitrogen treatmer t3 did not dif—
ferentially lower the carbohydrate res epves of common kentucky bluegrass
and Pennlawn creeping red fa ' ue. The glucose, fructose and sucrose
fractions vane affected little by nitrogen treatncnts. Temperature mav
have icon the dorizant iact or. Uligonaccharide other than sucrose
(PigUlE S and Appendix Table II), pros en t only in fierion and Toronto
Cfieening bentgrass leaves in appreciable co centrations, showed an in—
verse relationship between nitrogen t“eatments and Oligocaccharifle ccn~
centraticn. Under no treatuent did oligosacchavidc show much variation.
More extrere fluctuations were caused by other environwental factors.
Total. caruuuiflrdte values for Pennl1un (Appendix Tat-3.9 X) contained
a lag in peaks for the O and 3 treatments elative to E, 9 and 12 treat-
ments. Soil temperatures prior to June 8 were considerably below .0
degrees F. L01 1 org1n13 r.1s involved in organic decompociticn ani release

of nitrogen may h1ve retpo m1ed to the warmer soil temmn Iatunes arC‘nd June 8.

801m le nit“o en appliei noril 13, Ray 15 and Huy 31 in the b, 9 and 12

.3
.‘23
1+

tznatmente may have overcome thii .POgen 3101t1ce and stimulated plint

enzyme Syet;e m3 of produce carbohydrate. The 0 and 3 treetmcnts lacked

suf.‘ ic1er t soluble nitrogen to initiate this enzyme activity. Larncr tem—

peratures June 8 to 22 appeared to accomplish an effect similar to the

6, 9 and 12 treatments June 8. All treatments steadily dccreaeed in perm

cent total carbohydrate as soil tenpe: atures remained near 80 degrees F.
Results of the regrowth in t% e cark exveriment lackei the precision

and specificity of chemical determinations. The teln iency of values be-

tween treatments to overlap (lecreae zed their P lmi ilitv v. T1? ta relat mg

39

Percent of Dry “eight

Percent of Dry Weight

Figure 5. Oligosaccharide Minus Sucrose
Merton Kentucky Bluegrass

A.
3 r ______,0 Treatment
.. _. - _ 3 Treatment
— — 6 Treatment
— ----- 9 Treatment
___.__.12 Treatment
2 b .nnun.“511 Treatments

 

 

Hay ll May 23 June 8 June 22 July 8 July 22 Aug. 8 Aug. 22

Toronto Creeping Bentgraes

 

 

1
May 11 May 23 June a June 22 July 8 July 22 Aug. 3 Aug. 22

treatments to height lacked consistency. However, the lower nitrogen
treatrer ts produced the most ary matter. 1nl s agrees positively with

chemical indications that the lower nitrogen treatweuts generally contained
the maxim L173 reserve carbohyirates.

Total leaf cer uOH :jydr ate concentrations for all four grasses with
80 degree temperatures and the 12 pOind tr eatmer t only decreased to lev—
els one-quarter or greater of tnei r seasonal maximum. Tcte l carbohvdrates
did not do Cline to zero This 94.,e3‘o the possibility that carbohydrates
are not the only factor whic: mig?ht limit growth under high temperature
conditions. Meyer and Anders on' s (13) exhaus tiv e cove ge of tem era-
ture ~91 lent relationships exein;)lifies the oeuplexfi vof the problem . A
legical explanation could possibly be found by ccneideri1g the relation-
ships between teupereture and the reaction rate of n enzyme-catalyzed
reac ion. ”LufmJ-Citdljfed reaction rates are temperature d spen ient due
to temperature affecting the kinetic energies of the reactants and the
protein structure. teroera u as in the 80 F. range and higher
appearel to exert an adver3e effect on carbohydrate reserves of the four

cool seaeou grasses scuoiefl, and this effect exceeled t1 at attr 3b: itable

to the applied nitrOgen t1wetne.-s.

Ml

SUEEARY AND CONCLUSIONS

A technique for quantitative carbohydrate determinations using
densitometer readings from paper chromatograms was shown to be prac-
tical for analyzing large numbers of samples.

The greenhouse environment produced leaf tissue considerably lower

:osaccharide than were field samples.

I J

‘3

in oli
The polysaccharide present in all grasses studied was identified to

be a .gluCOpolyfructan and quantitatively analyzed by a simple
colorimetric test.

Effects attributable to nitrogen treatments were observed in the
oligosaccharide and polysaccharide fractions containing more than

two hexose moieties. Oligosaccharide concentrations greatly exceed-
ed fructosan under field conditions. The di- and monosaccharides

did not Show differential concentrations related to nitrogen treat~
ments.

The addition of more than one and one-half pounds of actual nitro-

gen per thousand square feet produced excess nitrogen conditions,

as evidenced by parallel carbohydrate responses.

Nitrogen differentials for an experiment in time should be applied
peri dicelly to weintain relatively constant treatnent differentials.
The technique of measuring plant food reserve as indicated by regrowth
in the dark, produced results which, although less specific, a,:eed

O

with chem.ca11y determined carbohydrate reserve values, particularly

rt-
:1”
(a)
O
H
[~4-
‘3
O

‘\

”saccharide ani polysaccharide concentrations.

8.

Temperatures in the 80 F. range and higher appeared to exert an
adverse effect on the carbohydrate reserves of the four cool season
grasses studied, and this effect exceeded that attributable to the
applied nitrogen treatments.

Under the conditions of this study and the nitrogen treatments used,
the total carbohydrate level in the leaves of the four grasses did
not appear to be Present in concentrations which were inadequate

for growth.

#3

l.

2.

3.

5.

5.

7.

8.

11.

LITERATURE CITED

Archbold, H. K. 1940. Fructosans in monocotyledons. A review.
New Phytol. 39:185-219.

Bacon, J. S. D. 1959. The trisaccharide fraction of some
monocotyledons. Biochem. J. 73:507~51u.

Brown, 5. M. 1943. Seasonal variations in the growth and chemical
composition of kentucky bluegrass. Missouri Exp. Stat. Bull.
380.

De Cugnac, A. 1931. Recherches sur les glucides des Graminees.
Ann. Sci. Nat. 13:1-129.

Dubois, M., Cilles, K. A., Hamilton, J. K., Ribers, P. A. and Smith,
Fred. 1956. Colo: unetric method for determination of sugars
and related substances. Anal. Chem. 28:350-356.

Ehara, Kaoru and Tameka, Shizeyuki. 1961. Effect of temperature on
the growth behavior and chemical composition of waii— and
cool- season ;?wra ses. Proc. Crop Sci. Soc. Japan. 29:304-306.

Giovannozz i-Sermanni, Giovanni. 195 6. A new solvent for quantita-
tive paper chromatography of sugars. Nature. 177:586—587.

Graber, L. F., Nelson, N. T., Leuke1,W. A. and A1bert, W. B. 1927.
Organic food reserves in relation to the growth of alfalfa
and other perennial herbaceous plants. Wise. Agr. Elxp. Stat.
Bull. 80.

Jansen, R. 6., Forbes, R. M. an5 Carlson, Don M.1958. A review of
the carbohydrate conawt tuants of routhagss. NCR Pub. 88.

Harrison, C. H. 1934. Response of kentucky bluegrass to variations
in temperature, light, cutting and fertilizing. Plant. Physiol.
9 3 83-106 0

Jordan, E. E. 1959. The efFect of environment a1 factors on the car—
bo1yi rate and nutrient levels of creeping bentgrass (“grostis

Palustri.). Purciue University. M. S. thesis (Unpub. 5.
Juska, F. J. and Hanson, A. A. 1981. Effects of interval and height

of mowing on growth of Marion and common kentucky cluesrass
(Poe pratencis L). Agron. J. 53:385-388.

v.14

13.

in.

15.

17.

p...
(O
O

19.

20.

Laidlaw, R. A. and Reid, 8. G. 1952. Analytical studies on the
carbohydrates of grasses and clovers. 1. Development of
methods for the estimation of the free sugar and fructosan
contents. J. Sci. Fd. Agric. 3:19—25.

Laidlaw, R. and Reid, 8. G. 1950. Filter paper chromatography:
Extraction of sugars from the paper at room temperature.
Nature 156:H76-H77.

Lopatec11,L. E., Longair, E. L. and Kastinn, R. 1962. Quantita-
tive ch11nges of solub.1e cazbo-.ydrates in stems of solid- and
hollow-s temmed wne its. Can. J. Bot. MO:1223¢1228.

Martin, A. J. P. and $7n.e, R. L. M. 19u1. A new form of
chromatogram employing two liquid phases. 1. A t.k o.‘y of
chromatography. Biochem. J. 35: 1358~1364.

McFarren, E. F., Brand, K., and Rutkowski, H. R. 1951. Quantita-
tive determination of sugars on filter paper chronatograms
by direct photometry. Anal. Chem. 23:1146-11u9.

McFary, W. L. and Slattery, M. C. 1945. The colorimetric deter-
mination of fructosan in plant material. J. Biol. Chem.
1572161-187.

Meyer, 8. S. and Anderson, D. B. 1952. Plant physiology. D. Van
Nostrand Co., Inc. Princeton, New Jersey.

Paech, K. and Tracey, M. V. 1955 a. Modern methods of plant analy~
sis. Zeiter Band vol. 2. Sp ringer—Verlag.

Sprague, V. C. and Sulliv “a. J. T. 19 50. Reserve carbohydrates in
orchard g mass clipp fie} peiiodical-ly. Plant Physiol. 25:92-102.

Sullivan, J. T. and Sprague, V. G. 19 49 he effect of tern erau
ture on the growth and compos Ht ion of the stubble and roots
of perennial ryegrass. Plant Physiol. 24: 706-718.

Troughton, A. 1957. The underground organs of herbage grasses.
Bull. No. nu. Commonwealth Bureau Pastures and Field Crops,
Marley, Berkshire.

APPENDIX

Table I. Leaf oligosaccharide percentages determined on a dry weight
basis for four grasses under five nitrogen treatments June 8 to August 22,
1363.

 

 

 

 

 

 

 

 

 

A Marlon kentucky bluegrass
June June July July Aug. Aug.
Treatfent 8 2? 8 22 8 22 Mean
0 3.86 6.37 6.28 5.63 4.54 4.43 2.60
3 3.47 5.56 5.66 5.04 4.48 4.51 2.39
6 .83 4.66 4.98 4.31 3.79 4.19 1.90
9 1.14 4.57 4.29 3.23 2.70 3.63 1.63
12 1.52 4.23 4.38 3.43 2.42 3.72 1.64
Mean 1.08 2.54 2.56 2.16 1.79 2.05
B Toronto creeping bentgrass
Treatwent
0 2.79 1.86 4.72 4.23 4.42 6.21 2.02
3 2.25 1.11 4.24 2.97 3.68 4.81 1.59
6 1.93 .14 3.85 2.56 2.89 4.31 1.31
8 1.61 0 3.41 2.36 ”.39 3.42 1.10
12 .51 0 3.13 2.34 2.03 3.34 1.04
Mean 1.02 .31 1.94 1.45 1.54 2.21
C Pennlawn creeping red fescue
TZ’V‘! fitment
0 1.88 4.38 4.92 3.50 2.76 4.52 3.66
3 1.88 3.56 3.42 4.08 2.64 4.08 3.28
6 1.78 3.28 3.22 3.52 2.94 3.28 3.00
9 2.20 3.22 3.16 3.42 3.40 3.20 3.10
12 3.34 2.86 2.94 3.50 2.92 3.50 3.18
Ream 2.22 3.46 3.53 3.60 3.13 3.72
D Common kentucky bluegrass
Treatnent
0 3.70 3.78 4.22 3.34 3.30 4.30 3.77
3 1.84 4.78 4.46 3.54 3.84 4.42 3.83
6 3.04 4.84 4.40 3.28 3.46 3.78 3.80
9 4.30 5.46 4.24 3.22 3.88 3.96 4.18
12 2.78 5.46 4.36 3.30 4.16 4.34 3.92
Mean 3.15 4.23 4.34 3.34 3.73 4.16

46

Table II.

Leaf Ollgosaccharide minus sucrose percentages determined

on a dry weight basis for four grasses under five nitrogen treatnents

June 8 to August 22, 1963.

 

 

A Eerion kentucky bluegrass
June June
Traatrent 8 92
O .19 .75
3 .08 .37
6 O .07
9 0 0
l2 0 0
Mean .09 .29
B Toronto creeping bentgrass
Treatment
0 .07 .66
3 .07 .34
6 O ..07
9 O 0
12 0 0
Mean .03 .21
; Pennlawn creeping red fescue

 

 

O O .69
3 0 .35
6 O O
9 O O
12 0 0
Mean 0 .21
D Common kentucky bluegrass
Treatwent
0 O .07
3 O .07
6 0 .07
9 0 .08
12 O .08
Kean 0 .07

July
8

 

1.12
.75
.37

0
0

.05

.80
.46
.13
.11
.05

.31

.70
.110

O

(J
R.)

.31
.31
.11
.08
.07

.18

07

July
22

 

1.12
.7H
.37
.06

.96

.67
.06

.01
.35

00000

 

1.06
.67
.26
.13

.06

00000

0

00000

O

 

1.0
.67
.3”
.10
.lu

.69
.21

00000

 

.70
.47
.21
.02
.01

.71
.38
.13
.06
.00

0
U!

0
MC.)

OOON

.06
.06
.03
.03
.03

APPENDIX

Table III. Analysis of variance for oligosaccharide concentrations
in the leaves of Toronto creeping bentgrass under five nitrogen treat~
ments, June 8 to August 22, 1963

Source BY MS
Replication (R) 1 .0052

N treatments (N) 9 1.9352 **
RN, E1 M .0094
Dates (D) 5 4.6011 **
RD, 22 5 .osus

ND 20 .0404**
END, E, x 20 .011u

** Significant at the 1 percent level

* Significant at the 5 percent level

Table IV. Analysis of variance for oligosaccharide concentrations
in the leaves of Marion kentucky bluegrass under five nitrogen treat-
ments, June 8 to August 22, 1963

Source MP MS
Replication (R) 1 .1540

N treatments (N) u 2.3u51 an
RN, Bl u .0398
Dates (D) 5 3.02u5 he
RD. 52 5 .1766 *
ND 20 .1200
END, 33 20 .0571

** Significant at the 1 percent level

* Significant at the 5 percent level

#8

Table V. Mean values for height (inches) and dry weight (grams)
for 1 irowth in oarkness of four grasses under five nitrOgen treatments,

Pounis nitrogen Toronto Pennlawn Herion Common
per thousand creeping creeping kentuck; lentucky
Silive feet A_ bentcrass red fescue bluegrass b1ue*rass

 

 

Heifiht Weight Height Weight Height weight Height Weight

 

 

0 2.63 .28 .63 .09 3.25 .37 2.“,

LC
0

b.)
O

h.)
o

1.;
u.)
o

H
N)

3 1.63 .32 3.38 .ou 3.75 .45

h)
o

0"
L.)
o

....
0'1

6 1.25 .33 1.13 .09 5.00 .MS
9 .63 .15 1.13 .01 9.38 .30 2.38 .12

12 1.25 .28 1.5 .02 3.38 .21 2.00 .14

Table VI. A comparis
extraction using a Soxhlet apparatus versu
arrangement.

-on of two methods for soluble carbohydrate
s a Buchner—flask-funnel

,.. , . T5. . m. 3 ‘-
F33‘Cr3flt O 1 LP] “8.1.3“:
Vethod Sucrose Glucose Fructose Fructosan

 

Mad“. I “mum

 

Trial A Buchner Flask 1.5 ~ .09 .115
Pea pretensis

 

Soxhlet App. 1.u1 - .08 .035

Trial B Buchner 1.22 - - .09
Pna‘pratensis

 

Soxhlet 1.08 - — .025

#9

Table VII. Leaf carbohydrate percentages determined on a dry weifiht
basis occurrinfi in fiarion kentuckv bluazraes under three single applica-
ti‘c’n nitr'of‘hn tmatw—ants, Ausfufgt 5, 1"}(‘3

 

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6 2.9“ .23 - 3.89 .“3 ".82
Aug. 8 O 2.97 .45 - 3.61 .5 n.53
6 2.28 .HE - 2.53 .37 3.59
12 2‘28 .2 ‘ 2.83 of ‘) Sans
A11". 10 O 2.? .21 - Qoffi .Fd 3-53
6 2.7g .31) * 2.7?) .12) 9.13
12 2.0-3 ‘36 - 7.90 .1? 3.38
Aug. 16 0 2.hw - — 3.58 .17 3.95
5 2.30 ~ ~ 2.33 .0 2.31
12 2.33 ‘ - 9.30 .01 2.31

50

a

Tania VIII. Ledf aflruohgirata pQICénta:és dutarL13ma on
baais occurring in com30n kantucky Liuaaragu u Jar tbrua gin E
tiun niaro cu treatzgnts, august 5, laws

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a ... - - 1.5% .c:

12 1.7a - - 1.7% .-1

flux. 13 3 1.43 .33 r

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51

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2:223:15; mmmring in 'I'r'mczrto crew-av. ir; 5.: barn: r333 w: imp
tion n?t?0'en trfiatrunt$, fiUfUSt 5, 1383.

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6
12

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