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ABSTRACT

CULTURAL AND ENVIRONMENTAL FACTORS INFLUENCING
THE STOMATAL DENSITY AND WATER USE RATE OF
PENNCROSS CREEPING BENTGRASS

BY

Robert C. Shearman

The effects of various cultural and environmental
factors on the stomatal density and water use rate of
Penncross creeping bentgrass were investigated. The fac-
tors included (a) light intensity, (b) temperature, (c)
cutting height, (d) soil moisture, (e) irrigation fre-
quency, and (f) nitrogen nutritional level. The relative
importance of these cultural and environmental factors in
determining the water use rate was determined.

Stomatal density counts were made from clear-
nitrocellulose replications of the leaf blade surface
using a light microscope at 430 magnifications. A special
wind tunnel apparatus was used to determine the water use
rate of the turves. The atmospheric drought-stress condi-
tions maintained in the chamber were 33 C, 40% relative
humidity, 4300 lux, and a constant wind velocity of 4 miles

per hour. The turves were exposed to these conditions for

Robert C. Shearman

a 12 hour period. The water use rate was determined from
the percent moisture lost during the exposure period.

Higher light intensities resulted in an increase
in stomatal density and water use rate. The stomatal den-
sity was reduced by 48% when the light intensity was re-
duced from 25,800 to 3762 lux, while the water use rate
declined by 50% for the same treatments. Suboptimal growth
temperatures resulted in a reduction in the stomatal den-
sity and water use rate. A 30% reduction in stomatal
density occurred between 20 and 10 C temperature while the
water use rate declined by 20%. No differences were found
in stomatal density and water use rate at the 20 and 33 C
growing temperature.

The water use rate increased with increased heights
of cut. Increasing the cutting height from 0.7 to 2.5 cm
resulted in a 53% greater water use rate. Turves cut at
12.5 cm had a water use rate which was double that at 0.7
cm. Increased nitrogen nutritional levels of 0.23, 0.91,
and 1.83 kg per 92.9 m2 resulted in reduced stomatal den-
sity and water use rate. The decline in water use rate at
the higher nitrogen nutrition levels was for a short—term
drought-stress period and not based on an entire growing
season.

The stomatal densities were inversely related to
the amount of water applied. The water use rates decreased

when the water application rate was increased from 1.3 to

Robert C. Shearman

10.0 cm per week. Infrequent irrigation resulted in a
reduction of the water use rate, while the stomatal den—
sity was increased. The water use rate was positively
correlated to the percent vegetative cover.

Light intensity, cutting height, and frequency of
irrigation had the greatest effect on the water use rate.
Nitrogen nutritional level was intermediate in its effect,-
while temperature and water application rate had the least
effect on the water use rate.

The stomatal densities from the various studies
ranged from 72 to 125 per square millimeter. A 31% in-
crease was noted between the second and sixth leaf blade
on the culm. A three fold increase in stomatal density
was found when the upper surface was compared to the lower

surface of the leaf blade.

CULTURAL AND ENVIRONMENTAL FACTORS INFLUENCING
THE STOMATAL DENSITY AND WATER USE RATE OF

PENNCROSS CREEPING BENTGRASS

BY

Robert C. Shearman

A THESIS

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

MASTER OF SCIENCE

Department of CrOp and Soil Sciences

1971

ACKNOWLEDGMENTS

The author would like to extend his sincere ap-
preciation to Dr. J. B. Beard for his guidance during the
course of this investigation and for his constructive
criticism in the preparation of this manuscript.

Appreciation is also expressed to Dr. P. E. Rieke
for his critical review of the manuscript and to the other
members of the guidance committee, Dr. S. N. Stevenson and
Dr. J. M. Vargas for their assistance and instruction.

A very special thanks is given to my wife, Linda,
for her understanding, love, and assistance during this

investigation.

ii

TABLE OF CONTENTS

Page
LIST OF TABLES o o o o o o o o o o o o o o o o o o 0 V
INTRODUCTION 0 O O I O O O O O O O O O O O O O O O O l
LITEMTURE REVIEW 0 O I O O I O O O O O O O C O O O O 3
The Water Use Rate of Turfgrasses . . . . . . . . . 3
Stomatal Relationships to Water Use Rate . . . . . 4
Effect of Light and Temperature on the Water
use Rate 0 I O O O ‘ O O O O I O O O O I O O O O O O 9

Effect of Clipping Height on the Wate Use Rate . . 12
Effect of Irrigation Frequency and Soil Moisture

on the Water Use Rate . . . . . . . . . . . . . . . 12
Effect of Turfgrass Nutrition on Water Use Rate . . l4

MATERIAL AND METHODS . . . . . . . . . . . . . . . . 17

Source of Penncross Creeping Bentgrass . . . . . 1?
Establishment Procedures . . . . . . . . . . . . 17
Cultural Practices . . . . . . . . . . . . . . . 18
Procedure for Stomatal Mount Preparation . . . . 19
Percent Moisture Loss Determination . . . . . . . 20
Description of Studies Conducted . . . . . . . . 23
Environmental Studies . . . . . . . . . . . . . . 23
Study I . . . . . . . . . . . . . . . . . . . . 23
Study II . . . . . . . . . . . . . . . . . . . 24
Cultural Studies . . . . . . . . . . . . . . . . 25
Study III . . . . . . . . . . . . . . . . . . . 25
Study IV . . . . . . . . . . . . . . . . . . . 26
Study V . . . . . . . . . . . . . . . . . . . . 27
Study VI . . . . . . . . . . . . . . . . . . . 28

RESULTS AND DISCUSSION . . . . . . . . . . . . . . . 31

Study 1. The Effects of Light Intensity on

Stomatal Density and Water Use Rate . . . . . . . . 31
Study 2. The Effects of Temperature on the

Stomatal Density and Water Use Rate . . . . . . . . 33
Study 3. The Effect of Cutting Height on Water

Use Rate . . . . . . . . . . . . . . . . . . . . . 35

iii

Page

Study 4. The Effect of Three Water Application

Rates on the Stomatal Density and Water Use Rate . 37
Study 5. The Effects of Three Irrigation

Frequencies on the Stomatal Density and Water

Use Rate . . . . . . . . . . . . . . . . . . . . . 39
Study 6. The Effect of Nitrogen Nutrition on

Stomatal Density and Water Use Rate . . . . . . . . 42
The Effect of Leaf Blade Surface and Position

on the Stomatal Density . . . . . . . . . . . . . . 46
The Relative Importance of Cultural and Environ-

mental Factors Influencing Stomatal Density and

Water Use Rate . . . . . . . . . . . . . . . . . . 48

CONCLUSIONS 0 O O O O O O O O O O O O O O O O O O O O 5 2

BIBLIOGMPHY O O O O O O I O O O O O I O O C O O O O 54

iv

LIST OF TABLES

Table Page

1. N-P-K nutrient levels for nutritional ex-
periment expressed as kilograms per 92.9
square meters . . . . . . . . . . . . . . . . 29

2. The effect of three light intensities on the
water use rate of Penncross creeping bent-
grass expressed as percent moisture lost
during a 12 hour exposure period at 33 C,
40% relative humidity, and 4300 lux . . . . . 31

3. The influence of three light intensities on
the average stomatal density for upper and
lower leaf surfaces of Penncross creeping
bentgrass expressed as number per mm . . . . 32

4. The effect of three temperature treatments on
the water use rate of Penncross creeping
bentgrass expressed as percent moisture
lost during a 12 hour exposure period at
33 C, 40% relative humidity and 4300 lux . . 34

5. The effect of three temperature treatments on
the average stomatal density for upper and
lower surfaces of Penncross creeping bent-
grass leaf blades expressed as number of
stomata per mm2 . . . . . . . . . . . . . . . 35

6. The effect of three heights of cut on the
water use rate of Penncross creeping bent-
grass expressed as percent moisture lost
during a 12 hour exposure period at 33 C,
40% relative humidity and 4300 lux . . . . . 36

7. The effect of three water application rates
on the water use rate, expressed as percent
moisture lost, of Penncross creeping bent-
grass during a 12 hour exposure period at
33 C, 40% relative humidity and 4300 lux . . 37

Table Page

8. The effect of three water application rates
on the average stomatal density for upper
and lower surfaces of Penncross creeping
bentgrass leaf blades expressed as number
of stomata per mm2 . . . . . . . . . . . . . 38

9. Visual estimates of the percent vegetative
cover for Penncross creeping bentgrass
turfs receiving three rates of water
application . . . . ... . . . . . . . . . . . 39

10. The effect of three irrigation frequencies on
the water use rate expressed as percent
moisture lost during a 12 hour exposure
period at 33 C, 40% relative humidity, and
4300 lux . . . . . . . . . . . . . . . . . . 40

11. Visual estimates of percent vegetative cover
for Penncross creeping bentgrass turfs re-
ceiving three frequencies of irrigation . . . 41

12. The effect of three irrigation frequencies on
the average stomatal density for upper and
lower surfaces of Penncross creeping bent-
grass leaves expressed as number of stomata
per mm . . . . . . . . . .p. . . . . . . . . 41

13. The effect of three nitrogen nutritional
levels on the water use rate expressed as
percent moisture lost during a 12 hour
exposure period at 33 C, 40% relative
humidity and 4300 lux . . . . . . . . . . . . 43

14. The effect of three nitrogen nutritional
levels on the average stomatal density
for upper and lower surfaces of Penncross
creeping bentgrass leaves expressed as
number of stomata per mm . . . . . . . . . . 44

15. Total of clipping weights recorded as grams
yielded above the 5 cm cutting height for
the dates of harvest of November 22, Decem-
ber 5, and December 19, 1970 . . . . . . . . 45

16. Shoot density counts recorded as shoots per
2.5 cm for Penncross creeping bentgrass
receiving three levels of nitrogen
nutrition . . . . . . . . . . . . . . . . . . 45

vi

Table

Page
17. Root organic matter production recorded as
grams of organic matter produced for Penn—
cross creeping bentgrass receiving three
levels of nitrogen nutrition . . . . . . . . 46
18.

The effect of leaf blade position and surface

on the stomatal d3nsity~expressed as number

of stomata per mm Penncross creeping
bentgrass . . . . .

O O O O O 47

vii

INTRODUCTION

Penncross creeping bentgrass (Agrostis palustris

 

Huds.) is one of the most widely used seeded grasses on
greens in the United States. It has a vigorous growth
habit and requires a high intensity culture that results
in a thatching tendency if not prOperly maintained. Cul—
tural practices for maintenance of a quality Penncross
turf generally include (a) high nitrogen fertilization,
(b) close, frequent mowing, (c) soil cultivation and mod—
ification, (d) preventative fungicide applications, and
(e) irrigation.

The creeping bentgrasses, such as Penncross, are
quite susceptible to drouth stress. The growth rate, in-
tensity of culture, and shallow root system under close
mowing necessitates frequent irrigation during moisture
stress periods. The amount and frequency of irrigation
required are determined by various cultural and environ-
mental factors. Factors affecting the rate of turfgrass
growth are probably the most influential in effecting the
water use rate. Included are light, temperature, soil
moisture, frequency of irrigation, cutting height and fre-

quency, and level of nitrOgen nutrition. The relative

importance of many of these cultural and environmental
factors on the water use rate of turfgrasses has not been
determined.

The objective of this investigation was to deter-
mine the relative importance of several cultural and envi-
ronmental factors on the water use rate of Penncross
creeping bentgrass. The factors studied were (a) light
intensity, (b) temperature, (c) soil moisture content, (d)
cutting height, (e) frequency of irrigation, and (f) level
of nitrogen nutrition. The influence of these factors on
stomatal density was also observed. Correlations between
stomatal density and water use rate were determined in

relation to the cultural or environmental factors studied.

LITERATURE REVIEW

The Water Use Rate of Turfgrasses

The consumptive use or water use rate is defined
as the total amount of water required for turfgrass
growth plus the quantity lost by transpiration and evap-
oration from soil and plant surfaces (3). It has been
demonstrated that the evapotranspiration rate of a turf is
greater than the evaporation rate from a bare soil (34,
35). The increase in amount of water lost from a turf is
due to transpirational losses from the extensive leaf sur-
face area exposed to the atmosphere plus the turfgrass
root system which increases the amount of water removed
from the soil profile. The water use rate for actively
growing turfgrasses can vary from 1.75 to 5.25 cm per week
depending on the environmental and cultural conditions and
may exceed 7.5 cm per week under high atmospheric moisture
stress (3).

A number of factors influence the water use rate
of turves including (a) length of the growing season, (b)
evapotranspiration rate, (c) rate of growth, (d) the turf-
grass species or cultivar, (e) intensity of culture, (f)

amount of traffic, (g) soil texture, (h) precipitation,

and (i) available soil moisture (3). The total amount of
water used per year is partially determined by the length
of the growing season. The amount of water used may also
vary due to factors influencing the evapotranspiration rate
and growth rate throughout the growing season. Both are
influenced by the turfgrass species or cultivar, intensity
of culture, soil texture, amount of soil moisture, precip-
itation, and intensity of traffic.

The factors noted for influencing the water use
rate such as the species or cultivar, intensity of culture,
precipitation, amount of soil moisture, and light intensity
have been noted by various workers to have marked influence

on stomatal density (9, 13, 14, 21, 22, 23, 24, 25, 26).

Stomatal Relationships to Water Use Rate

Approximately 90 percent of the water lost from a
leaf occurs by diffusion through the stomata. Factors
influencing the stomatal number and fluctuation in stomatal
aperature provide the indirect mechanism for regulation of
the transpiration rate (18). The response of stomata to
light, carbon dioxide concentration, and internal water
stress are predictable (18, 31). Light and water stress
are the major factors determining stomatal aperature. The
light effect has been explained quantitatively by a photo-
synthetic reduction-of the CO2 concentration in the inter-

cellular spaces and guard cells (18).

Stomata are essentially closed in the dark. A
minimal light intensity is necessary for stomatal opening

which varies with the species. Tomatoes (Lycgpersicum

 

esculentum Mill) have been reported to require at least

2956 lux, while 968 lux causes opening in Pelargonium s2,

 

(18). The degree of stomatal opening increases with in-
creased light intensity up to a maximum intensity for the
particular species where a maximum stomatal aperature
occurs. Very high light intensities may cause a decrease
in stomatal opening (18, 30). This could be an indirect
effect attributed to high leaf temperatures or a high rate
of water loss which causes a tissue water stress.

The water balance within the plant is influenced
by the interrelated processes of absorption, translocation
and transpiration. Changes in water content or hydrature
of the leaf are generally expressed as changes in water
deficit. The water deficit is considered a very powerful
stomatal regulator. When above a critical level, the
water deficit will override all other stomatal opening
stimuli (3, 18).

In general, studies concerning the temperature
influence on stomata have been difficult to evaluate. It
is hard to separate the effects of temperature on the (a)
diffusion coefficient, (b) water deficit of the leaf tis-
sue, and (c) atmospheric relative humidity. Within the

normal-range of temperatures, 10-25 C for cool-season

turfgrasses, temperature has little effect on the steady-
state stomatal aperature. Temperatures higher than 30-35 C
have a closing effect which may contribute to the cause of
midday closure of stomata (18). Temperature does not in-
fluence differentiation of stomata, but can produce morpho-
logical abnormalities in the stomata (1). Carbon dioxide
levels increase in the intercellular spaces with increased
temperature (18). This response is thought to be due to
increased respiration.

Stomata play a vital role in gas exchange between
the leaf and the adjacent atmosphere. They function as
the primary pathway for water vapor, carbon dioxide, and
oxygen exchange (31). Stomata may occur on both surfaces
of a leaf (an amphistomatic leaf) or on one surface only,
either the upper (an epistomatic leaf) or the lower (a
hypostomatic leaf) (16). Although stomata may occur on
both leaf surfaces, they are more commonly found on the
lower surface of most plants (16, 27, 31). However, this
does not seem to be the case for grasses. Several re-
searchers have reported a higher stomatal density on the
upper as opposed to the lower surface of such species as

blue panicgrass (Panicum antidolate Retz), corn (Zea Mays

 

L.), barley (Hordeum vulgare L.), creeping bentgrass

 

(Agrostis palustris Huds.), and annual bluegrass (Poa

 

annua L.) (13, 27, 23, 24). Other workers have made sim-

ilar observations for such species as alfalfa (Medicago

sativa L.), beans (Phaseolus vulgaris L.), pumpkin

 

(Cucurbita peps L.), and tomatoes (Lycopersicum esculentum

 

 

Mill.) (10, 16, 27).

Generally speaking, the frequency of stomata may
range from 50 to 500 per square millimeter (31). The
stomata may be separated by no more than 1 or 2 epidermal
cells at the higher frequency. Creeping bentgrass and
annual bluegrass were reported to have a frequency of l
stomata to 2 epidermal cells. The same species grown
under infrequent watering had a l to 4 ratio, indicating
that variability within a species could be caused by ex-
posure to various environmental and cultural factors (23).
The stomata are arranged in parallel rows in species of
monocotyledons which have veined leaves. The stomata tend
to be scattered in species with netted-veination.

Stomatal density can vary with the leaf position

 

and plant maturity. Blue panicgrass (Panicum antidolate
Retz.) was-shown to have fewer stomata per unit area in
the younger leaves at the tOp of the culm than leaves col-
lected at either the middle or base of the culm (13).
Preliminary studies with the same plant showed 30 percent
fewer stomata at the seedling stage of growth than at the
time of pollination. Work with several cultivars of a1-
falfa indicated an inverse relationship between stomatal
density and leaf position (10). Leaves sampled from the

apex of the plant had significantly more stomata per mm2

than those from the base. Samples from all cultivars had
significantly greater stomatal densities on the adaxial
leaf surface compared to the abaxial surface. Stomatal
frequencies were studied on the lower leaf surface of 649
cultivars from the World Collection of Barley (24). Fre-
quencies decreased progressively from the flag to the lower
leaves, with the flag~leaf having approximately twice the
stomatal density of the basal leaves. Stomatal frequencies
ranged from 36 to 98 per mm2 with a mean of 64 on the flag
leaf of the cultivars observed. A negative correlation

was obtained between the stomatal frequency and size. The
researchers suggested that the negative correlation between
frequency and size of stomata might be the result of a
compensatory relationship, such that the total pore area

of different cultivars is approximately equal.

Although stomatal density varies significantly for
most species with position and surface of the leaf, work
with blue panicgrass indicated that the numbers do not
vary significantly as to position on the same leaf. Im-
pressions at the base, middle, and apex of the leaf aver-
aged 116, 119, and 119 per mmz, respectively (13).

Stomatal density varies significantly among culti-
vars within a species, as well as between species. Among
cultivars of alfalfa studied, Ladak maintained the greatest
number of stomata regardless of the surface and Sonora the

lowest (10). Stomatal densities were significantly

different among clones of blue panicgrass (l4). Clones
selected for high forage production under irrigated condi—
tions had a significantly higher stomatal density than
clones which were selected for seedling drought tolerance.
A significant negative correlation between drought toler-
ance and mean stomatal density was found, indicating that
clones which had the highest number of stomata per unit

area were the least drought tolerant seedlings.

Effect of Light and Temperature on
the Water Use Rate

 

The role of light in influencing the water use
rate of turfgrass can be closely associated with its var-
ious effects on the stomata, leaf and shoot density, root
growth, cuticle development, and carbohydrate reserve.

The water content of plant tissues varies diurn-
ally. The water content is greater during the night, with
a maximum in the early morning hours and decreases during
the day with a minimum at midday. The osmotic pressure‘
within the tissue varies inversely with the degree of hy-
dration. Increased osmotic pressure during the daylight
hours is caused by (a) the buildup of soluble carbohydrates
and other organic compounds resulting from photosynthesis,
and (b) a decrease in the degree of hydration due to water
loss through transpiration exceeding water absorption by

\
the root system (3). The response of stomatal aperature

10

to light and temperature have already been discussed in
the previous section, as well as, the importance of the
stomata in water loss by transpiration.

Increased light intensity and duration of illumi-
nation play roles in increasing the number of stomatal
initials and the stomatal frequency (28, 30). An increase
in epidermal cell size and a decrease in size of stomata

were also noted. Alfalfa (Medicago sativa L.) and birds-

 

foot trefoil (Lotus corniculatus L.) were reported to have
a significantly higher number of stomata when grown in
full-sunlight of approximately 96750 lux compared to plants
grown under similar conditions but shaded by 92% Saran

Shade Cloth (11). Leaves of silverrod (Solidagg_virgaurea

 

R.) were also found to have greater numbers of stomata per
unit area when grown in the sun as opposed to leaves grown
in the shade (4). Miskin reported a similar response in
barley with increased light intensity, while temperature
was reported to have very little effect on stomatal fre-
quency (24). In addition to the effect of increased light
intensity on stomatal frequency and size of epidermal
cells, cuticle thickness and consistency is also influenced
due to the effect of light on the oxidation and condensa-
tion of fatty acids involved in the process of cuticle
formation (20). Low light intensities tend to result in a
thin cuticle, reduced root system, and low carbohydrate

reserves. Investigation with Coastal bermudagrass (Cynodon

ll

dactylon L.) showed that reduced light intensity resulted
in a decrease in percentage of plant weight associated
with the roots and rhizomes (7). Carbohydrate reserves
were also reduced.

Turfgrasses have an optimum temperature range for
growth and development. For most of the cool-season turf-
grass species, the optimum is in the soil temperature
range of 15 to 20 C. Plants exposed to temperatures above
or below their growth optimum are subject to decreased
growth. The greater the deviation from the Optimum temp-
erature, the greater is the chance that growth will cease
and death occur. Temperatures which encourage more rapid
shoot growth rates tend to increase the water use rate.
Temperatures within the 10-25 C range have been reported
to have little effect on stomatal aperature, while higher
temperatures of 30-35 C have a closing effect (18). Stu-

dies with sunflower (Helianthus annus L.) plants have

 

shown an increase in transpiration with increased soil
temperatures (33). The water use increases with tempera-
tures up to a maximum. The decrease in the water use rate
at higher temperatures is associated with stomatal closure,
and with the fact that root permeability to water is de-

creased at the higher soil temperatures.

12

Effect of Clipping Height on the
Water Use Rate

 

 

The water use rate is thought to increase with the
increase in height of the grass plant. This is associated
with the increase in total leaf area exposed to desiccating
conditions and with the increase in the extent of the root
system. There has been very little reported regarding the
effect of cutting height on the water use rate of turf-
grasses.

The evapotranspiration rates for perennial ryegrass

(Lolium perenne L.) and white clover (Trifolium repens L.)

 

were greater when the plants were allowed to grow to 25-30
cm, than when they were clipped at 2.5-5.0 cm (25). Work-
ers using Kentucky bluegrass turves clipped at various
levels reported that soil water extraction was directly
proportional to the height of cut (21). These workers
assumed that water extraction from the soil was directly

proportional to the number of absorbing roots.

Effect of Irrigation Frequency and Soil
Moisture on the Water Use Rate

 

The turfgrass plant absorbs water from the soil
through the root system. Water is absorbed primarily
through the root hairs which are located just above the
root tips. The root hair zone varies in magnitude with
species, environmental conditions existing during devel-

Opment, and the age of the root (3).

13

Frequent, light irrigations reduce drought toler-
ance due to a decrease in the extent of the root system
(23, 28). Increased soil moisture has also been reported
to decrease the number of root hairs, and the associated
absorptive capability of the root system. Creeping bent-
grass and annual bluegrass watered 2 and 6 times a week
for a period of 5 weeks were more prone to wilting with
the more frequent irrigation treatment (23). Roots from
plants in the flats watered 6 times per week were somewhat
shorter than those less frequently watered. However, as
'soil moisture stress increases water use efficiency, dry
matter production and transpiration of the grass plants
decrease. Clones of blue panicgrass were observed to be
more efficient in water use when soil moisture levels were
maintained near field capacity (14).

Investigations with soybeans (Glycine max L.)

 

showed that as soil moisture was reduced below field capa-
city the evapotranspiration was reduced. Soybeans grown
under drought and nondrought soil conditions had different
evapotranspiration rates. The ratio of nondroughted to
droughted was 0.71 (16).

Soil moisture levels have various indirect effects
on the plant, which are associated with the water use rate.

Sunflower (Helianthus annus L.) and common smartweed

 

(Polygonum hydropiper L.) had larger but fewer stomata per

 

unit area when grown under dry soil conditions. Also, the

14

leaves were wider and thicker (28). A reduction in water
loss from soybeans grown under droughty conditions was
associated with an accumulation of lipids on the leaf sur-
face (9). These workers found that water loss from
droughted plants continued to decrease even after the
stomata were observed to close. This decrease in water
loss was associated with increased cuticle thickness. The
cutin layer in annual bluegrass and creeping bentgrass was
found to be almost nonexistent on plants maintained under
excessive moisture levels (23).

Leaves of frequently watered grasses have more air
space between cells of the mesophyll tissue than those of
infrequently watered plants (23, 28). The addition of
fertilizer was noted to add to this increase in air space
between cells of the mesophyll tissue. Grasses grown
under semi-arid soil conditions had very little air space
between the cells. This variation in air space is import-
ant. The increase in air space between cells allows for
more surface area from which the water may evaporate and

be lost as water vapor through the stomata and cuticle.

Effect of Turfgrasstutrition on
Water Use Rate

 

Factors which have a direct effect on the shoot
growth rate of the turfgrass plant will also have a direct

effect on the water use rate. Turfgrass nutrition plays

15

an important role in the amount of moisture extracted from
the soil by the plant. Nitrogen fertilization has been
reported to increase the water use rate of turfgrasses (3,
5, 8, 12, 19, 22, 32). Early reports from literature re-
viewed before 1915 indicated that the water requirements
of plants was reduced by the use of fertilizers, and that
the reduction was greater on poor soils, than on fertile
soils (5).

Studies were conducted in Israel concerning the
influence of irrigation frequency and nitrogen fertiliza-

tion on the water use rate of kikiyugrass (Pennisetum

 

clandestinum Hochst.), a turfgrass species. The objective
was to find a combination of nitrogen fertilization and
irrigation practices which would maintain desirable turf-
grass quality, but require a minimal amount of water. The
conclusions were that turfgrasses receiving nitrogen ap-

plications of 2.1 kg. per 1000 m2

per month (1.4 pounds of
nitrogen per 1000 sq. ft. per month) and irrigated every

25 days did not produce any greater yield than nonferti-
lized plants irrigated every 7 days (22). The regular use
of nitrogen did not produce excessive growth but maintained
adequate color and shoot density when water was not applied
at too high a frequency. They also concluded that less
frequent irrigation only conserved water. Nitrogen ferti-

lization of irrigated grasses increased the evapotranspi-

ration rate (19). Studies-with alfalfa, Kentucky bluegrass,

16

and quackgrass (AgroEyron repens Beaun) resulted in similar
findings (12, 32). Growing conditions that prevent rapid
plant growth and promote the accumulation of dry matter
would have a greater ability to withstand drouth. Rhizomes
of quackgrass from fertilized soil were found to be more
severely injured by drought than those from unfertilized
soil (12). Studies with various turfgrass species, such

as Kentucky bluegrass, Canada bluegrass (Poa compressa L.),

 

rough bluegrass (Poa trivialis L.), annual bluegrass, and

 

colonial bentgrass (Agrostis tenuis L.) indicated that
plants grown at high nitrogen levels were less able to
withstand soil drought than those grown at low nitrogen
levels (8).

The water use efficiency is reported to increase
when the level of nitrogen nutrition is increased (19, 32).
This is the result of a decrease in the amount of water
required to produce a unit of dry matter. Sprague reported
that the water requirement per unit of dry matter produced
is increased when growth is limited by nitrogen fertiliza-
tion or a reduction in stored reserves (32). The water
use rate is of greater consequence for turfgrasses than

the efficiency for dry matter production.

MATERIAL AND METHODS

Source of Penncross Creeping
Bentgrass

 

The investigations were conducted with Penncross
creeping bentgrass (Agrostis palustris Huds.) using the
same certified seed lot (Lot No. HQ-69216l) throughout all
studies. The seed test of the lot (April, 1969) showed a
purity of 98.47 percent and a germination of 94 percent.

There were no other crops or weed seeds present.

Establishment Procedures

The turves were established in 0.473 liter (16 oz.)
wax cottage-cheese containers. A soil mixture of 80%
white, silica sand and 20% sandy loam topsoil was used in
all studies except those involving the influence of nu-
trient levels, where only silica sand was used. The con—
tainers were filled with the soil mixture to within 1.25
cm of the rim allowing an 8.75 cm growing medium. The
surface area of the containers was 99.4 square centimeters.
Drainage was provided by holes punched in the bottom of
the wax containers.

The seed was spread uniformly across the soil sur-
face at a rate of approximately 0.228 kg. per 92.9 m2, and

was covered with a thin, 0.6 cm layer of a sand and soil

l7

18

mixture. Several layers of cheese cloth were placed on

the soil surface to maintain favorable moisture conditions
during establishment. Each study was seeded 10 days prior
to initiation of the various treatments. The establishment
period under an automatic mist-irrigation system in the
greenhouse was similar for all the plants. Temperature

and light conditions were not monitored during establish-
ment. The plants were 1.88 cm in height when the experi-

mental treatments were initiated.

Cultural Practices

 

Similar cultural practices such as cutting height,
fertilization, and irrigation were utilized for each study.
Variations in cultural practices will be discussed with
each study where appropriate. The cutting height was
maintained at 5.0 cm with the turves being clipped at
weekly intervals. The cutting height study was the only
exception to this procedure.

The nutrient solution used was a modified Hoagland's
solution (17), having an N-P-K ratio of 4:1:2. It was ad-
justed to a pH of 7.0 by the addition of sodium hydroxide.
The nutrient solution application rate approximated 0.912
kg. of nitroqen per 92.9 m2 per month. Nutrient solution
applications were made twice weekly. All the plants were
watered daily with the exception of the watering frequency

study. Those studies involving the influence of fertility,

l9

irrigation frequency, and amount of water applied were the
only exceptions.

The two basic types of data collected in all stu-
dies were stomatal density and water use rate. The pro-
cedures for preparation of stomatal mounts and determina-
tion of water use rate were standard throughout all

studies.

Procedure for Stomatal Mount
Preparation '

 

Leaves for stomatal impressions were randomly
chosen from the same position on the culm of the plants
in each treatment container. They were selected at a
similar stage of development, the sixth leaf. The method
for making stomatal impressions was similar to that re-
ported by Sampson (29) with the following modifications.
The silicone rubber monomer procedure was eliminated. Im-
pressions were made directly from the surface of each leaf
using a nitrocellulose preparation (clear fingernail
polish). This gave a satisfactory negative print of the
turfgrass leaf surface.

Each leaf blade was firmly attached to a sheet of
Parafilm with a clear cellulose adhesive taped to the leaf
base and tip. The nitrocellulose preparation was spread
evenly across the leaf surface and allowed to dry for 6

hours. After drying, the impressions were ready for

20

peeling. The tapes applied to the base and tip of each
leaf acted as tabs which aided in peeling the transparent
replica from the leaf blade.

The transparent replica was then transferred to a
polished microscope slide and placed under a slip cover.
The edges were firmly bound with clear cellulose adhesive
tape. Transfers were made swiftly to prevent the speci-
men's replica from curling. Curled, clouded, or otherwise
undesirable replications were discarded.

Stomatal density counts were made at 430 magnifi-
cations. The actual area observed was 0.159 square milli-
meters, but counts were converted to numbers per mmz.
Counts were based on the following considerations: (a)
all counts were made from the margin of the leaf toward
the mid-vein; (b) they were taken from the middle of the
leaf; (c) density counts were made on the abaxial and ad-
axial surfaces; and (d) any stomata within or touching the
field were included in the density count. The number of
replications for the stomatal density counts varied with

the individual study, but were within the range of 4 to 10.

Percent Moisture Loss Determination

Wet weight determinations were made 12 hours after
the containers were watered to saturation. The 12 hour
drainage period was adequate to approach field capacity,

for the sandy soil mixtures used in the investigations.

21

Field capacity is achieved when there is no longer any
rapid downward movement of water through the soil (6).'
Moisture movement continues to take place but at a much
slower rate. The movement is due primarily to capillary
forces effective in the micropores. The macrOpores would
greatly exceed the micrOpores in the sandy soil mixture
used, thereby decreasing the amount of time necessary to
obtain a moisture level simulating field capacity.

The total moisture at the beginning of each study
ranged from 20 to 33%. The percent moisture was constant
within each study. Once the steady state of moisture per-
centage was approached, the containers were readied for
exposure to the desiccating conditions of the wind tunnel.
Preparation for the wind tunnel involved covering the
bottoms of the containers with polyethylene to prevent
water losses through the drainage holes, and recording wet
weights prior to the exposure period.

The containers were then placed in a special wind
tunnel apparatus with a controlled environment of (a) 33 C,
(b) 40% relative humidity, (c) 4300 lux light intensity
and (d) a constant air-flow of 4 miles per hour. These
conditions were chosen after some preliminary experiments
in order to maximize the water use rate and thus minimize
the duration of the exposure period. The latter was im-
portant in experiments involving large numbers of replica-

tions. The exposure period selected was 12 hours. This

22

was ample time for marked differences in the water use
rates to occur between treatments.

The containers were placed in the wind tunnel ap-
paratus on a rotating platform that allowed all containers
to be uniformly exposed to the environmental conditions
within the chamber. The relative humidity and temperature
within the chamber were monitored with a Hygrodynamics
hygrometer (Model no. 15-3001) that was accurate to 11.5%.
A light source was supplied from overhead fluorescent
lighting and an additional incandescent light source
placed immediately above the chamber.

The containers were again weighed at the termina-
tion of the 12 hour exposure period, and the weights re-
corded (termination weight). The oven dry weights were
then determined by placing the turves in a 105 C forced-
air drying oven for 24 hours. Initial studies showed that
containers exposed to the same conditions for 36 and 48
hours had the same oven dry weight as those exposed for 24
hours. The percent moisture lost was calculated as
follows:

Wet Weight-Termination Weight x 100 = Percent Moisture
Wet Weight-Oven Dry Weight Lost

 

Wet weight minus the termination weight was used to deli-
neate the amount of moisture lost during exposure to con—

ditions of the wind tunnel apparatus. The total moisture

23

content was the difference between the wet weight and the
oven dry weight. The percent moisture lost was determined
by dividing the total moisture content into the amount of
moisture lost during the exposure period and multiplying
by 100. These calculations were made on each observation

within the individual studies.

Description of Studies Conducted

Six individual studies were conducted during the
course of this investigation. The environmental and cul-
tural factors investigated in terms of their influence on
stomatal density and water use rate were: (a) light in—
tensity, (b) temperature, (c) amount of water applied, (d)
frequency of watering, (e) cutting height and (f) nitrogen

fertilization.

Environmental Studies

 

Study I.--The effect of light intensity on stomatal
density and water use rate.

Three light regimes were used in this study to
determine the effect of light intensity on the stomatal
density and water use rate of Penncross creeping bentgrass.
The light intensity treatments were (a) 3762 lux, (b)
25,800 lux, and (c) sunlight. Each treatment had 10 rep-
lications totaling 30 observations for the study. The

experimental design was completely randomized.

24

Those turves under light intensities of 3762 and
25,800 lux were placed in separate, environmentally con-
trolled growth chambers set at a constant day-night temp-
erature of 20 C and a 16 hour photoperiod. Those exposed
to full sunlight were placed in an Open area near the
greenhouse where the light intensities ranged from 29,025
to 96,750 lux. They were exposed to varying day-night
temperatures with an average daily temperature range of
15-20 C and a photoperiod of approximately 16 hours.

The preconditioning period was terminated after 3
months. Leaf samples for stomatal measurements were taken
at this time.' The water use rate was determined by the
use of the wind tunnel apparatus and determining the per-
cent moisture lost by the method previously_described.

The data was appropriately analyzed, and the correlation
between stomatal density and water use rate determined.

Study II.--Determining the effect of temperature
on stomatal density and water use rate.

The influence of temperature on stomatal density
and water use rate of Penncross creeping bentgrass was
evaluated in this study. The three temperatures used were
10, 20, and 33 C with eight replications per treatment
totaling 24 observations. The treatment temperatures were
based on soil temperatures which were monitored by soil
probe thermometers. The three controlled environment

growth chambers used had 12 hour photOperiods with a

25

21,500 lux light intensity. Each temperature treatment

was held constant throughout the preconditioning period.
Upon termination of the 2.5 month treatment period,

the stomatal density and water use rate were determined

for each growing temperature treatment using the procedures

previously outlined. The correlation coefficient was cal-

culated for stomatal density and water use rate.

Cultural Studies

Study III.--The effect of cutting height on the

 

water use rate of grasses.

The effect of three cutting heights on the water
use rate of Penncross creeping bentgrass was evaluated in
this study. The cutting heights were 0.7, 2.5, and 12.5
centimeters. They were replicated 4 times giving a total
of 12 observations for the study.

The establishment procedure was the same as that
described in the general discussion. After establishment
the plants were maintained in a controlled environment
growth chamber, having a 12 hour photoperiod of 21,500 lux
and a constant day-night temperature of 20 C. The cultural
practices were the same as those previously described,
with the exception of the cutting height treatments.
Clipping treatments were begun when the plants reached
3.75 centimeters and were continued throughout the study.

Plants were clipped weekly, except those at the 12.5

26

centimeter cutting height which were not clipped during
the preconditioning period.

The study was terminated after 3 months and the
plants exposed to the standard desiccating conditions of
the wind tunnel apparatus. The percent moisture loss as
influenced by the treatments was determined according to
the procedure previously described.

Study IV.--Determination of the effect of water
application rate on stomatal density and water use rates.

The influence of water application rate on stomatal
density and water use rate was evaluated in this study.
The study was established by the methods previously de—
scribed. The turves were placed in a controlled environ-
ment growth chamber with a constant day-night temperature
of 20 C and a 12 hour photoperiod of 21,500 lux. The
three treatments were 1.3, 2.5 and 10.0 centimeters of
water applied per week. It was determined that 210 milli-
liters applied weekly were equivalent to 2.5 centimeters
of water applied per 99.4 cm2 per week. Therefore, 15,
30, and 120 milliliters per day were applied for each of
the three treatments. Preliminary tests demonstrated that
an application of 120 milliliters of water was necessary
to obtain drainage from the container. The treatments
were replicated six times giving a total of 18 observa-

tions for the study.

27

Cultural practices were similar to those mentioned
in the general discussion, with modifications in the ap-
plication of the nutrient solution. The nutrient solution
was applied as growth and color dictated. It was applied
to all the containers at the 15 milliliter application
rate. Additional water was used to adjust the differences
in application rates for the 30 and 120 milliliter
treatments.-

The turves were sampled for stomatal density counts
after 3 months of treatment. They were then placed in the
special wind tunnel apparatus and the water use rate was
determined.

Study V.--The effect of irrigation frequency on
stomatal density-and water use rate.

The irrigation frequency study was established and
maintained under similar conditions to those for the water
application rate study. The treatments consisted of (a)
watering only when visual wilt occurred, (b) watering 3
times per week to saturation, and (c) watering daily to
saturation.. There were 9 replications of each treatment
for a total of 27 observations. Samples were taken for
stomatal density data at the end of the 3 month treatment
period. The plants were then exposed to the standard wind
tunnel conditions for the purpose of determining water use

rates.

28

Study VI.--Determination of the effect of nitrogen
on stomatal density and water use rate.

The stomatal density and water use rate were de-
termined for plants of Penncross creeping bentgrass which
had been subjected to three levels of nitrogen nutrition
(Table 1). All the treatments received the same micro-
nutrient levels which were applied in concentrations sim-
ilar to those used in a complete Hoagland's nutrient solu-
tion (17)..

The nutrient solution was applied daily throughout
the 3 month treatment period. The growing conditions,
cutting height and frequency were the same as those pre-
viously described in the cultural practice section. Sto-
matal density counts were taken when the treatment period
was terminated. The turves were exposed to the standard
conditions of the wind tunnel after the stomatal density
samples were taken. The correlation coefficient was de-
termined for the relationship between the stomatal density
and the water use rate.

An auxiliary study was conducted, under the same
establishment procedures, to determine the clipping weight
production, root organic matter production, and shoot den-
sity for the same nitrogen nutritional levels. Clipping
production was determined on a fresh weight basis. The
root organic matter production was determined by ashing

the roots in a muffle furnace at 700 C for 4 hours. Shoot

29

 

 

 

 

 

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Imam oooa Ame a.~m Imam coca Ame m.~a Imam coca Ame m.~m mumnenz
\.mnHV \mxv \.mnHV \mxe \.mgae \mxv pcosummue
M m z

Hm>oq ucowuusz

 

 

mamnmoHHx mm commmnmxm ucoEHnmmxo HMGOADHHUSC

.mnouma ohmsvm m.mm mom
MOM mam>oa ucmwuusc Mrmrz .H magma

3O

densities were determined from a grid system with 2.5 cm2
units. The densities were determined from an average of
three counts per pot.

Initial soil moisture content was determined for
each study. The soil moisture content varied between 18—
33% for the various studies. No significant variation in

initial soil moisture content was noted between treatments

of the same study.

RESULTS AND DISCUSSION

Study 1. The_Effects of Light Intensity on
Stomatal DenSity and Water Use Rate

The effect of light intensity on the water use

rate of Penncross creeping bentgrass is shown in Table 2.
The three treatments were 3762 lux, 25,800 lux and full
sunlight. Water use rates expressed as percent moisture
loss were 26.3, 39.1, and 46.1, respectively for the three
treatments. Table 3 shows the results for stomatal densi-
ties obtained from the same study. The stomatal densities
were 78.6, 113.2, and 125.8 stomata per mm2 for the three
treatments. The water use rate was positively correlated
to the stomatal density (0.88).
Table 2. The effect of three light intensities on the

water_use rate of Penncross creeping bentgrass

expressed as percent moisture lost during a 12

hour exposure period at 33 C, 40% relative hu-
midity, and 4300 lux.

 

 

Percent Moisture Lost

 

Light Intensity (average for Multiple
Treatments (lux) 10 replications) Comparison Test
3,762 26.3 a
25,800 39.1 b
sunlight 46.1 c
S; = 1.22

 

Values with the same letter are not significantly
different at the 1% level (Duncan's Multiple Range Test).

31

32

Table 3. The influence of three light intensities on the
average stomatal density for upper and lower
leaf surfaces of Penncross creeping bentgrass
expressed as number per mm .

 

 

Stomatal Number

 

per mm
Light Intensity (average for Multiple
Treatments (lux) lO replications) Comparison Test
3,762 78.6 a
25,800 113.2 b
sunlight 125.8 c
s- - 0.31

 

Values with the same letter are not significantly
different at the 1% level (Duncan's Multiple Range Test).

Previous research with dicotyledons showed that
increased light intensity and duration of illumination
play a role in increasing the number of stomatal initials
therefore, increasing the stomatal density (28, 30). The
results of this experiment showed a similar response. The
increased stomatal density resulting from higher light
intensities was associated with an increase in the water
use rate.

Light intensity also affects other plant responses
which could have influenced the water use rates observed
in this study. The total leaf area and shoot growth were
reduced at the lowest light intensity compared to plants

grown at the two higher intensities. Bermudagrass leaves,

33

shoots, and root numbers were reduced at lower light in-
tensities in a previous study (7). A reduction in leaf
area exposed to desiccating conditions is usually asso-
ciated with a decrease in transpiration. The reduced root
production observed at low light intensities could also

limit the water absorbing capabilities of the grass plant.

Study 2. The Effects of Temperature on the
Stomatal Density and Water Use Rate

 

Light intensity and water deficit are major factors
determining stomatal aperature. Since the greatest portion
of water lost by the plant is through the stomata these
are very important factors influencing the water use rate
of a plant. The temperature influence on the water use
rate of plants is not as clearly defined. Little effect
on stomatal aperature has been reported within the normal
temperature range for cool season turfgrasses (18). Temp-
eratures higher than 30-35 C tend to have a closing effect
on the stomata (18, 33). There has been considerable dif-
ficulty in attempting to evaluate the influence of tempera-
ture because of the confounding influence on the diffusion
coefficient, the water deficit of leaves,.and the relative
humidity. These problems were minimized by the controlled
wind tunnel technique used in these investigations.

Three temperature treatments, 10, 20, and 33 C,

were included in this experiment. The water use rates

34

were 51.9, 61.9, and 61.3 percent for the respective
treatments (Table 4). Plants grown at 10 C had a substan-
tially reduced water use rate compared to the 20 and 33 C
treatments. There was no difference in the water use
rates between plants grown at 20 and 33 C. The effect of
these same three temperatures on stomatal density are
shown in Table 5. The 10 C treatment had a stomatal den-
sity of 72.3 per mm2. The stomatal density of the 10 C
treatment was significantly different from the 20 and 33 C
temperature treatments. There was no difference in sto-

matal density at the 20 and 33 C temperatures.

Table 4. The effect of three temperature treatments on
the water use rate of Penncross creeping bent-
grass expressed as percent moisture lost during
a 12 hour exposure period at 33 C, 40% relative
humidity and 4300 lux.

 

 

 

Temperature Percent Moisture Lost
Treatments (average for Multiple
(centigrade) 8 replications) Comparison Test
10 51.9 a
20 61.9 b
33 61.3 b
s- = 2.79
x

 

Values with the same letter are not significantly
different at the 1% level (Duncan's Multiple Range Test).

35

Table 5. The effect of three temperature treatments on
the average stomatal density for upper and lower
surfaces of Penncross creeping bentgrass leaf
blades expressed as number of stomata per mm .

 

 

 

Temperature Stomatal Number per mm2
Treatment (average for Multiple
(centigrade) 8 replications) Comparison Test
10 72.3 a
20 99.4 b
33 103.1 b
s- = 3.57
x

 

Values with the same letter are not significantly
different at the 5% level (Duncan's Multiple Range Test).

There was no significant correlation between sto—
matal density and water use rate. The correlation coeffi-
cient was 0.46. However, water use rate and stomatal den-

sity both increased between 10 and 20 C.

Studyg3. The Effect of Cutting Height
on Water Use Rate

 

It is generally thought that higher cutting
heights result in an increased water use rate of turf-
grasses, but little is known regarding the magnitude of
this relationship. The effects of 0.7, 2.5, and 12.5 cm
cutting heights on the water use rate of Penncross creep-
ing bentgrass were investigated in this study. The re-

sults of this study are shown in Table 6. The water use

36

rate expressed as percent moisture lost, increased as the

cutting height increased (Table 6).

Table 6. The effect of three heights of cut on the water
use rate of Penncross creeping bentgrass ex-
pressed as percent moisture lost during a 12
hour exposure period at 33 C, 40% relative
humidity and 4300 lux.

 

Height of Percent Moisture Lost

 

Cut Treatment (average for - Multiple
(cm) 4 replications) Comparison Test
0.7 21.4 a
2.5 32.8 b
12.5 45.0 c
S- = 1048
x

 

Values with the same letter are not significantly
different at the 1% level (Duncan's Multiple Range Test).

Raising the cutting height from 0.7 to 2.5 cm re-
sulted in approximately a 50% increase in the water use
rate. Higher heights of cut encourage more extensive and
deeper rooting (3). The leaf area exposed to desiccating
conditions is greater at the higher cutting heights. This
would enhance the extent of water loss by transpiration.
Also, the increase in extent of the root system would
allow the plant to draw moisture from a greater portion

of the soil profile.

37

Study 4. The Effect of Three Water Application
Rates on the Stomatal Density and
Water Use Rate

 

Three application rates of water, 1.3, 2.5 and

10.0 centimeters, were applied weekly to Penncross creep-
ing bentgrass turves. Significant differences in water
use rates were recorded between those plants receiving 1.3
cm and 10.0 cm of water per week (Table 7). Plants re-
ceiving 2.5 cm had a water use rate which was not signif-
icantly different from the plants receiving either the
lowest or the highest water application treatments. There
was a trend toward decreasing water use rate between the
plants receiving 1.3 and 10.0 cm water application rates
per week.
Table 7. The effect of three water application rates on

the water use rate, expressed as percent moist-

ure lost, of Penncross creeping bentgrass during

a 12 hour exposure period at 33 C, 40% relative
humidity and 4300 lux.

 

 

Percent Moisture Lost

 

Water Application (average for Multiple
Rate (cm/week) 6 replications) Comparison Test
10.0 47.2 a
2.5 50.6 ab
1.3 55.0 b
s- = 1.68
x

 

Values with the same letter are not significantly
different at the 5% level (Duncan's Multiple Range Test).

38

The-stomatal density was inversely related to (a)
the amount of water applied weekly and (b) the water use
rates (Table 8). No significant correlation (0.55) was
found between the stomatal density and the water use rate.
Plants receiving 1.3 cm of water per week had significantly
fewer stomata per unit area than those receiving 2.5 and
10.0 cm. There was no difference between the latter two
treatments. A trend of decreasing stomatal density with
decreasing soil moisture was observed. Visual estimates
of vegetative cover were made for each treatment and rep-
lication in this study. The vegetative cover was signif-
icantly decreased at the lowest moisture treatment (Table
9). Visual observations of depth and extent of rooting
indicated an increase at the lowest water application rate.
Table 8. The effect of three water application rates on

the average stomatal density for upper and lower

surfaces of Penncross creeping bentgrass leaf
blades expressed as number of stomata per mm .

 

 

Number of Stomata

 

Water Application per mm2 (average for Multiple
Rates (cm/week) 6 replications) Comparison Test
10.0 120.1 a
2.5 117.6 a
1.3 102.5 b
s- = 4.40
x

 

Values with the same letter are not significantly
different at the 5% level (Duncan's Multiple Range Test).

39

Table 9. Visual estimates of the percent vegetative cover
for Penncross creeping bentgrass turfs receiving
three rates-of water application.

 

 

 

Water Percent Vegetative Cover
Application (average for Multiple:
Rates (cm/week) 6 replications) Comparison Test
10.0 95.8 a
2.5 88.3 a
1.3 51.7 b
s- = 3.14
x

 

Values with the same letter are not significantly
different at the 5% level (Duncan's Multiple Range Test).

Study 5. The Effects of Three Irrigation
Frequencies on the Stomatal Density
and Water Use Rate

 

The effects of three irrigation frequencies, (a)
watered only when wilt appeared, (b) watered 3 times per
week, and (c) watered 7 times per week, on the stomatal
density and water use rate of Penncross creeping bentgrass
were investigated. The pots were watered until the soil
was saturated at each watering. Plants watered only when
wilt appeared had a reduced water use rate compared to
those irrigated 3 and 7 times per week (Table 10). The
latter two treatments were not significantly different.

The decline in water use rate was positively cor-
related (0.99) with the decrease in the vegetative cover

(Table 11). A 47.8% reduction in vegetative cover was

40

noted for those turves which were watered only when wilt
was apparent. This was significantly different from the
16.1 and 7.2 percent reduction recorded for the irrigation
frequencies 3 and 7 times per week. There was no signif-
icant reduction in vegetative cover between the 3 and 7
times-per week irrigation frequency. A reduction in the
extent of rooting was also noted in the turves receiving
the least frequent irrigation treatments. The more fre-
quently irrigated plants appeared to have developed the
most extensive root system. The plants receiving 3 and 7
irrigations per week had the fewest stomata (Table 12).
Plants watered only when wilt appeared had a significantly
greater number of stomata than the more frequently irri-
gated treatments. Leaves of those plants watered only at
wilt were much narrower and reduced in area.
Table 10. The effect of three irrigation frequencies on
the water use rate expressed as percent moist-

ure lost during a 12 hour exposure period at
33 C, 40% relative humidity, and 4300 lux.

 

 

 

Irrigation
Frequency Percent Moisture Lost
Treatments (average for Multiple
(times/week) 9 replications) Comparison Test
At wilt 35.1 a
3 49.3 b
7 49.7 b

s- = 1.14
x

 

Values with the same letter are not significantly
different at the 1% level (Duncan's Multiple Range Test).

41

Table 11. Visual estimates of percent vegetative cover
for Penncross creeping bentgrass turfs receiv-
ing three frequencies of irrigation.

 

 

 

Irrigation
Frequency Percent Vegetative Cover
Treatments (average for Multiple
(times/week) 9 replications) Comparison Test
At wilt 52.2 a
3 83.9 b
7 92.8 b

s- = 3.48
x

 

Values with the same letter are not significantly
different at the 5% level (Duncan's Multiple Range Test).

Table 12. The effect of three irrigation frequencies on
the average stomatal density for upper and
lower surfaces of Penncross creeping bentgrass
leaves expressed as number of stomata per mm .

 

 

Irrigation

 

Frequency Stomatal Number per mm2
Treatments (average for Multiple
(times/week) 9 replications) Comparison Test
At wilt 117.6 a
3 90.6 b
7 93.7 b

s- = 2.68
x

 

Values with the same letter are not significantly
different at the 5% level (Duncan's Multiple Range Test).

42

There was no significant correlation between the
water use rate and the stomatal density. However, the
correlation between percent vegetative cover and water use
rate was significant (0.98). Turves receiving irrigation
only at wilt had a significant reduction in percent vege-
tative cover compared to those receiving 3 and 7 irriga-
tions per week. The decline in water use rate was asso-
ciated with the low shoot density of the turf maintained
at this irrigation frequency. Although the water use rate
was reduced, the loss of turfgrass quality at this irriga-

tion frequency was undesirable.

Study6. The Effect of NitrOgen Nutrition
on Stomatal Density and Water Use Rate

The nitrogen treatments utilized in this experiment
were (a) 0.23, (b) 0.91, and (c) 1.83 kg. per 92.9 m2
(0.5, 2.0, and 4.0 pounds of actual nitrogen per 1000 sq.
ft.) per growing month. The corresponding nitrogen-phos-
phorus-potassium ratios were as follows: (a) 1:1:2, (b)
4:1:2, and (c) 8:1:2. Turves grown under these nitrogen
levels were exposed to a stress period of 12 hours in
which the conditions were 33 C, 40% relative humidity, and
4300 lux. The percent moisture losses were 46.2, 40.2,
and 34.0 for the respective treatments, indicating a re-

duction in water use rate with increased nitrogen levels

(Table 13).

43

Table 13. The effect of three nitrogen nutritional levels
on the water use rate expressed as percent
moisture lost during a 12 hour exposure period
at 33 C, 40% relative humidity and 4300 lux.

 

 

 

Percent
Level of Nitrogen Moisture Lost Multiple
Nutrition2 (average for Comparison
(klg./92.9 m ) 6 replications) Test
.228 (0.5#/1000 sq. ft.) 46.2 a
.912 (2.0#/1000 sq. ft.) 40.2 b
1.825 (4.0#/1000 sq. ft.) 34.0 c

s- = 1.18
x

 

Values with the same letter are not significantly
different at the 1% level (Duncan's Multiple Range Test).

The stomatal density decreased with increased
fertilization (Table 14). Plants receiving the 1.83
treatment had a significantly_lower stomatal number per
mm2 than those receiving 0.23 kg. The average width of
the leaf at the midportion increased at the higher-N-
treatments. The leaf widths recorded were (a) 1.58, (b)
2.96, and (c) 3.03 mm for the three nitrogen nutritional
levels. The stomatal density increased as the leaf width
decreased. There was a positive correlation (0.60) between
the stomatal density and water use rates of the turves
studied.

Water use rate has been reported to increase with

an increase in the rate of nitrogen fertilization (3, 5,

44

8, 12, 19, 22, 32). The increase in water use rate, in
such cases, has been associated with an increased growth
rate and dry matter production. However, the water use
becomes more efficient on the basis of the amount of water
necessary to produce a pound of dry matter, when nitrogen
fertilization is increased.
Table 14. The effect of three nitrogen nutritional levels
on the average stomatal density for upper and

lower surfaces of Penncross creeping bentgrass
leaves expressed as number of stomata per mm .

 

 

Stomatal Number

 

Levels of Nitrogen per mm2 Multiple
Nutrition. (average for Comparison
(kg/92.9 m2) 6 replications) Test
.228 (0.5#/1000 sq. ft.) 88.1 a
.912 (2.0#/1000 sq. ft.) 83.6 ab
1.825 (4.0#/1000 sq. ft.) 78.6 b

 

Values with the same letter are not significantly
different at the 5% level (Duncan's Multiple Range Test).

The clipping weight production, shoot density, and
root organic matter production were observed for the three
nitrogen levels in an auxiliary study. The average fresh
weight of clippings did increase with increased nitrogen.
The fresh weights were 1.34, 2.84, and 2.48 gm for the
respective treatments (Table 15). A reduction in fresh

weight of clippings was noted for the 0.23 kg treatment.

45

However, no difference was noted between the 0.91 and

1.83 kg nitrogen levels. Shoot densities increased with

increased nitrogen nutrition (Table 16).

Table 15. Total of clipping weights recorded as grams'
yielded above the 5 cm cutting height for the

dates of harvest of November 22, December 5,
and December 19, 1970.

 

 

 

Clipping
Level of Nitrogen Weights Multiple
Treatment Nutrition (average for Comparison
Numbers' (kg/92.9 m2) 3 replications) Test
1 0.23 1.34 a
2 0.91 2.84 b
3 1.83 2.48 b
s; = 0.48

 

Values with the same letter are not significantly
different at the 5% level (Duncan's Multiple Range Test).

Table 16. Shoot density counts recorded as shoots per 2.5
cm2 for Penncross creeping bentgrass receiving
three levels of nitrogen nutrition.

 

 

Level of Nitrogen Shoot Density Multiple

 

Treatment NutritioB (average for Comparison
Numbers (kg/92.9 m ) 4 replications) Test
0.23 32.5 a
0.91 51.0 b
1.83 50.6 b
s- = 0.41
x

 

Values with the same letter are not significantly
different at the 5% level (Duncan's Multiple Range Test).

46

The lower nitrogen rate.promoted the development
of an extensive root system (Table 17), while it discour-
aged dry matter production and shoot density. The root
organic matter production at the 0.23 kg nitrogen level
was nearly 2 times greater than that at the two higher
nitrOgen levels. The increase in root organic matter
production and stomatal density associated with this nu-
tritional level would tend to lend support to these
findings.

Table 17. Root organic matter production recorded as
grams of organic matter produced for Penncross

creeping bentgrass receiving three levels of
nitrogen nutrition.

 

 

 

Level of Root Organic Matter
Nitrogen Production in Grams Multiple
Treatment Nutrition2 (average for Comparison
Numbers (kg/92.9 m ) 4 replications) Test
1 0.23 1.59 a
2 0.91 0.69 b
3 1.83 0.90 b

 

Values with the same letter are not significantly
different at the 5% level (Duncan's Multiple Range Test).

The Effect of Leaf Blade Surface and Position.
on the Stomatal Densigy

 

The stomatal density of the upper and lower leaf

surfaces of Penncross creeping bentgrass were compared at

47

each of two leaf positions (Table 18). The leaf positions
compared were the second and sixth leaf from the base of
the culm. Stomatal densities differed significantly be-
tween surfaces at-both leaf positions. The upper surface
had a greater number of stomata per mm2 in each case. The
sixth leaf had the greatest number of stomata per-unit

area for both surfaces when compared to the second leaf

 

 

position.

Table 18. The effect of leaf blade position and surface
on the stomatal density expressed as number of
stomata per mm Penncross creeping bentgrass.

Leaf Blade

Position Average for Multiple

and Surface 18 Replications Comparison Test

 

sixth upper 123.9 3
sixth lower 44.7 b a
second upper 90.6 a
second lower 37.7 b
*surface s; = 1.99
= 2.22

fposition s;

 

Values with the same letter are not significantly
different at the 1% level (Duncan's Multiple Range Test).

The average stomatal density for the lower compared
to the upper leaf position was significantly reduced, in-

dicating a greater number of stomata on the younger leaves.

48

Similar studies with blue panicgrass indicated that younger

leaves at the top of the
area than leaves collect
(13). It was also shown
were reduced at the time
seedling stage. Studies

showed that the stomatal

culm had fewer stomata per unit
at the middle or base of the culm
that the total number of stomata
of pollination as compared to the
with various cultivars of barley

density decreased progressively

from the flag leaf to the lower leaves (24). It would

appear that the relationship between stomatal density and

leaf blade position are not consistent among the grasses

and may vary between species.

The stomatal density for blue panicgrass did not

vary significantly in reference to position on the same

leaf blade (13). Counts were determined to be similar

whether taken at the base, middle, or tip of the leaf.

Similar results were obtained in this study. However,

all counts were taken from the middle of the leaf blade.

The stomatal density may

plants in general (31).

vary from 50 to 500 per mm2 for

The stomatal densities from the

various studies within this investigation ranged from 72

to 125 per square millimeter.

The Relative Importance of Cultural and Environmental
Factors InfluencingStomataI Density
and Water Use Rate

 

Reductions in light intensity, cutting height, and

frequency of irrigation resulted in the most significant

49

influence on the water use rate. Turves growing at 3762
lux had a 50% reduction in water use rate compared to
turves grown at 25,800 lux. The light intensity of 3762
lux is near the compensation point reported for many turf-
grasses (3). Turves clipped at 2.5 cm had a 53% greater
water use rate than those cut at 0.7 cm. The water use
rate doubled between turves cut at 0.7 cm and 12.5 cm.
Turves receiving irrigation only when wilt occurred had a
water use rate that was reduced 43% compared to those re-
ceiving irrigations 3 and 7 times per week.

The most significant factor influencing the sto-
matal density in this investigation was light intensity.
Literature previously cited indicated that light intensity
and duration of illumination play a role in increasing the
stomatal density (28, 30). Soil moisture-content,.irriga-
tion frequency, and temperature were intermediate in their
influence on the stomatal densities. The nitrogen nutri-
tional level had the least significant effect on stomatal
density of the factors studied.

The relationships between stomatal-density and
water use rate were investigated. High light intensities
increased the stomatal density which was positively corre-
lated (r = 0.88) with an increase in water use rate. In-
frequent irrigations resulted in an increased stomatal
density. However, the water use rate was inversely related

to the stomatal density and was significantly lower at the

50

least frequent irrigation treatment. The decline in water
use rate was positively correlated (r = 0.98) to a reduc-
tion in percent vegetative cover. This increase in sto-
matal density at the least frequent irrigation treatment
probably resulted in an increased water use rate on a per
plant basis; however, this increase was overshadowed by
the reduction in percent vegetative cover.

The nitrogen nutritional level was intermediate in
its effect on the water use rate, but had the least sig-
nificant effect on the stomatal density of all the factors
investigated. Nitrogen influenced other factors within.
the plant. The degree of hydration is increased with in-
creased levels of nitrogen fertilization (19, 32). The
increased tissue hydration results inra decreased water
deficit in relation to the microenvironment surrounding
the leaf blade. The changes in water content or hydrature
are expressed as changes in water deficit. The water
deficit is considered a very powerful stomatal regulator
which may override all other stomatal opening stimuli (3,
18). In addition to increasing the degree of tissue hy-
dration, the nitrogen nutritional level increases air
space between the cells of the mesophyll. The increased
air space between cells allows for more surface area from
which water may evaporate and be lost as water vapor the

stomata and cuticle.

51

The temperature and water application rate had the
least effect on the water use rate. A reduction in water
use rate of 20% occurred with decreased temperature. An
18% reduction occurred with increased rates of water ap-
plication. The stomatal density was reduced by 30% for
the temperature treatments and 20% with the water applica-
tion rates.

These studies indicate that cultural factors such
as cutting height, irrigation frequency, and nitrogen
fertilization have the greatest influence on reducing the
water use rate of turfgrasses. -Manipulation of one factor
within a maintenance program may not be significant. How-
ever, a combination of cultural factors could significantly
reduce the water demanded by a turf. Manipulation of
these factors where irrigation applications are limited in
respect to water source, capacity and facilities could be
very important for maintenance of desired turfgrass qual-
ity. Light intensity was the environmental factor that
had the greatest influence on the water use rate. Adjust-
ment of irrigation practices to the existing light inten-
sity-conditions, such as shade versus open sunlight and
cloudy versus sunny days is important in maintaining

quality turf.

CONCLUSIONS

The following conclusions can be made regarding

the influence of various cultural and environmental factors

on the stomatal density and water use rate of Penncross

creeping bentgrass:

1.

Stomatal density and water use rate increase with
increasing light intensity.

Growing temperatures below the Optimum for shoot
growth cause a reduction of stomatal number and
water use rate.

The water use rate increases as the cutting height
is increased.

The water use rate is reduced with infrequent ir-
rigation. However, a critical moisture balance is
necessary to maintain desirable turfgrass quality.
Stomatal density and root organic matter production
are reduced at higher nitrogen nutritional levels.
Clipping yield and shoot density are increased as
the level of nitrogen nutrition increases.

Water use rate declines with increased levels of
nitrogen nutrition for a short term atmospheric,

drought-stress period of 12 hours.

52

53

Stomatal density is influenced by leaf blade sur-
face and position. The stomatal numbers are
greater on the upper than on the lower surface of
the leaf blade. Stomatal density is reduced on
mature leaves when compared to younger leaves

located higher on the culm.

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