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i f/ o UNIVERSITY OF
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Bradenton GCREC Research Report
BRA-1994-19 (November 1994)
STOMATAL LIQUID INFILTRATION (SLI) TEST, CROP CULTURE,
AND AIR POLLUTION EFFECTS
S. S. WOLTZ
GCREC Research Report BRA1994-19
STOMATAL LIQUID INFILTRATION (SLI) TEST. CROP CULTURE,
AND AIR POLLUTION EFFECTS
S. S. Woltz'
Gulf Coast Research and Education Center
University of Florida, IFAS
5007 60th Street East
Bradenton, FL 34203
Stomates may be described as the breathing pores on the surface of the leaf that
permit the uptake of large amounts of carbon dioxide (C02) from a low
concentration in the atmosphere (0.033% by volume) without losing excess water
vapor when the plant is under moisture deficiency stress. They open and close
in a way that controls water vapor loss and expedites the uptake of C02, usually
when photosynthesis is actively converting the C02 to sugars. Water loss control
comes first, by the mechanism of action of the stomates. Stomate size, density
of occurrence on leaf surfaces, and the presence or absence from leaf surfaces
all vary with plant type and environment (20, 34, 35). Many plants, especially
woody plants, have no stomates on the upper leaf surface.
Foliar spray and stomates: Nutrient sprays, especially in combination with very
effective wetting agents such as the organo-silicones, are taken up better where
there are open stomates (4, 13, 16) and can occasionally be, relatively
ineffective if stomatal access is not available. The same rules apply to the
promotion of uptake internally of systemic pesticides, depending also on the
capacity for penetration of the cuticular membrane layer (6, 10, 13, 22, 24,27).
Phytotoxicity of spray applications may be altered by the condition of the
stomates and the leaf surfaces at time of spraying. If there are chances of leaf
injury due to the borderline safety of a spray material and rate used, then one
could avoid enhanced uptake by spraying when stomates are relatively closed (5,
7, 8, 28, 29, 30).
Environmental interactions: The distribution and action of the stomates are
closely related to the ambient environmental conditions of sunlight, wind,
humidity, time of day, temperature, and free moisture (5, 7, 8, 11, 33).
Irrigation timing and frequency programming may be supplemented by stomatal
measurements that act as a final test for how the top of the plant is faring in
water supply in conditions of root damage, salty soils (including excess
fertilizer salts), and low atmospheric humidity. Atmospheric pollutants may open
or close stomates and will gain entry at a much more rapid rate through stomates
that are fully open (3, 9, 14, 34) in contrast with those that are closed. Air
pollution damage may be related in history of occurrence to a sequence of events
associated with opening and closing of stomates in response to sunlight, climate
and pollutant. For this reason, studies of air pollution problems would well be
2The capable technical assistance of Patricia Jones is gratefully acknowledged.
supplemented by a correlated history of stomatal reactions (3, 9, 14). The
design of experiments on atmospheric pollution problems and handling of plant
material should consider stomatal action in test plants as well as field plants.
Test plants growing in containers and placed in an area of suspected atmospheric
pollution are often subjected to water stress and stomate closure more than the
field plants and may therefore not serve as good indicators of the presence of
pollutants. This problem may be reduced in severity by good water control in the
containers coupled with comparative checking of stomate apertures of field and
Diseases, and plant breeding: Stomates constitute a portal for entry of
pathogens into the leaf (2, 11, 12, 19, 21, 23, 25, 31). The plant and the
pathogen have a conflict over controlling the process, which plant breeding and
selection may mediate. Toxins produced by pathogens can cause the stomates to
remain open (28, 29) which could be a factor in disease. Crop productivity and
adaptation to environmental conditions are related to stomatal action. Poor
stomate control can result in wilty, water-inefficient plant material or reduce
the range of geographical usefulness. Photosynthetic efficiency is also linked
to stomatal performance. These considerations can be of importance in a breeding
program. Fortunately, most plant material by natural selection has become more
or less adapted to living with the environment and to a lesser degree with plant
Molisch (18), in 1912, evaluated the condition of stomates opened or closed -
by the use of an infiltrating liquid which revealed infiltration by a water-
soaked appearance under the leaf epidermis. Since that time, others have used the
infiltration procedure to estimate the degree of openness of stomates (1, 15, 26,
33). Application of the method has been limited, however, by the lack of the
desired capacity to quantify and reproduce data for measurements between workers
using different penetrating liquids. Methods more amenable to data collection
include the autoporometer that measures air flow through the leaf, humidity
measurement at the leaf surface, silicone imprints of stomates that are examined
under the microscope, and direct microscopic counts of stomates that are open,
with consideration for the degree of aperture. Liquid infiltration has compared
favorably with other methods in a semi-quantitative sense but lacks the desired
definition. There appear, however, to be many cases wherein the use of liquid
infiltration can play a special role to supplement other methods or substitute
for them in some practical applications.
The effectiveness of the stomatal liquid infiltration (SLI) method depends on the
selection of liquids (solutions, mixtures of liquids) that will penetrate the
stomates on the plant material of interest (1, 15, 26, 33). Features of the
liquid mixtures that make them useful include a workable degree of affinity for
the leaf surface and an ability to penetrate the leaf pores. Controlling
characteristics of penetration capacity include 1) sufficiently low surface
tension, 2) workable ability to wet the surface cuticle of the leaf and 3)
viscosity that is low enough to physically permit the liquid to pass through the
stomate. Liquid floods the sub-stomatal cavity giving a visible sub-surface
wetting making the leaf somewhat translucent and having the appearance of being
internally water-soaked. The method does not work on all plant material, however,
for various reasons such as visualization problems.
A semi-empirical approach to methods development is useful. Since the physical-
chemical nature of the leaf surface is not known and the details of solvents and
solutions are also only partially understood, a trial and error procedure is
needed to pair penetrating solutions with specific plant types.
This work explores methods of stomatal aperture estimation by SLI using a variety
of liquids on different types of plant material for applications indicated in the
Materials and Methods
The degree of stomatal opening was estimated by placing a drop of penetrant
solution on the upper or lower surface of the leaf and estimating the time
required for significant liquid infiltration. When about one-third of the area
under the liquid developed a water-soaked appearance it was considered to have
been significantly infiltrated and time (limit 10 seconds) was recorded. For
practicality, the use of a stop watch was replaced by timing estimates made by
counting numbers for an estimate of time using the procedure that suited the
observer as checked by a stop watch.
Infiltrating liquids tested included an array representing a wide range in
polarity, lipophilic nature, surface tension, viscosity, and surfactant qualities
(wetting agents). These liquids included mineral spirits, mineral oil, ethanol,
isopropanol, myristic acid, organo-silicone and other wetting agents, and water.
Many combinations of liquids as well as individual liquids may be effective, with
varied application to different types of plant material; for this reason, the
selection of liquids for use in this work covered a wide range of types of
Results and Discussion
A comparison of stomatal liquid infiltration rates for bean and cabbage (Table
1) reveals that as the amount of mineral oil used in a mixture of mineral spirits
and mineral oil is increased, the rate of infiltration rapidly declines. This
reduction in rate of infiltration is related to increased viscosity. A graded
series such as that in Table 1 is useful to establish an information base on the
stomatal condition of suitable leaves with a rapid, simple estimation. The
mixture selected depends on the plant material and the objectives in testing
stomates for degree of aperture, as discussed in the literature review. The 91%
isopropanol could be used on some plants but is probably not as generally
applicable because of the partially polar nature of the compound that would
interfere with spreading and penetration on leaf surfaces of a more completely
In Table 2, a comparison is shown of a wide variety of plants with a single
infiltrating liquid mixture. This specific mixture was used for screening
purposes, but may lack adequate penetrating speed and affinity for bean, pepper,
and Viburnum. More detailed observation and comparisons under varied
environmental conditions would be desirable. Part of any final choice should be
based on more definitive methods discussed in preceding sections.
An assortment of potential infiltrating solutions was tested on 6 plant species.
The most outstanding observation to be made from the data in Table 3 is that
there is a wide range of types of liquids that may be satisfactorily used for
this purpose. Here again, as in the preceding table, it is evident that the
specific results with combinations of plant material and infiltrating solution
have to be worked out according to the objectives. The upper surfaces usually
have less liquid infiltration than the lower surfaces of leaves when they both
have stomates. Zinnia stomates are the most conductive to liquid infiltration
which is in agreement with observations on higher sensitivity to foliar spray
toxicity observed in a number of experiments.
Summary: It may be said that the stomatal liquid infiltration (SLI) method of
evaluation of stomatal aperture holds benefits for many applications, such as
with spraying crops, studying air pollution, and as an adjunct to irrigation
practices. The main factors are that this method is inexpensive, immediately
available, and gives prompt indications on degree of stomatal action and presence
of functional stomates. Individuals attempting to apply the SLI method to their
specific plant-environment situations should use a trial procedure to determine
whether SLI could be of assistance. A first choice for infiltrating solutions
would be various percentage mixtures of mineral spirits and mineral oil as shown
in Table 1.
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stomatal opening as illustrated with coffee. Plant Phys. 29:97-98.
Bald, J. G. 1952. Stomatal droplets and the penetration of leaves by plant
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Black, V. J., and M. H. Unsworth. 1980. Stomatal responses to sulfur dioxide and
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Canny, M. J. 1990. Fine veins of dicotyledonous leaves as sites for enrichment
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Croft, P. J., M. D. Shulman, and R. Avissar. 1993. Cranberry stomatal
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Freer-Smith, and G. Taylor. 1992. Comparative evaluation of the effects of
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Field, R. J., and N. C. Bishop. 1988. Promotion of stomatal infiltration of
glyphosate by an organosilicone surfactant reduces the critical rainfall
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Hildebrand, D. C., and B. Riddle. 1971. Influence of environmental conditions on
reactions induced by infiltration of bacteria into plant leaves.
Johnson, J. 1944. Bacterial invasion through stomata. Phytopath. 34:1005.
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Munnecke, D. E., and P. Chandler. 1957. A leaf spot of philodendron related to
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Ormrod, D. J., and A. J. Renney. 1968. A survey of weed leaf stomata and
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Table 1. Comparison of stomatal liquid infiltration rates with various
Time of day 0830 hrs 1115 hrs 0830 hrs 1115 hrs
Side of Leaf Lower Upper Lower Upper Lower Upper Lower Upper
-- seconds --
100% MSz 1.0 1.3 1.0 1.0 1.0 1.0 1.0 1.0
80% MS 1.0 2.0 1.0 1.0 1.0 1.0 1.0 1.0
60% MS 1.7 2.7 1.0 2.0 1.0 2.0 1.0 1.0
40% MS 2.7 7.0 2.0 7.0 1.0 2.0 1.0 2.7
20% Ms 7.3 6.7 3.3 6.0 2.7 4.0 2.0 3.3
0% MS 10.0 10.0 6.7 8.7 8.0 8.0 6.7 9.7
91% IPY 2.0 9.7 1.3 2.0 1.0 1.0 1.0 1.0
z% MS= % by volume of mineral spirits (odor-free); remainder made to 100%,
pharmaceutical grade mineral oil.
YIP= Pharmaceutical grade 91% isopropanol.
xTime, seconds for at least 30% infiltration, average for 3 plants.
Table 2. Comparison of stomatal liquid infiltration ratesy for several types
of plants using a mixture of 60% mineral spirits and 40% mineral oil
at 3 times of day.
Time of day 0915 hrs 1115 hrs 1600 hrs
Side of Leaf Lower Upper Lower Upper Lower Upper
Crop -- seconds --
Bean 10.0 10.0 7.7 7.3 10.0 8.7
Cabbage 2.3 8.3 1.3 4.0 2.3 5.3
Pentas 1.7 10.0 2.0 10.0 4.7 10.0
Pepper 9.7 10.0 10.0 10.0 10.0 10.0
Sour Orange 4.0 z 4.7 5.0 -
Tomato Sdlg. 2.0 2.0 2.3 6.0 5.3 7.7
Tomato 2.3 4.0 5.0 8.7 5.3 10.0
Viburnum 8.0 -z 10.0 10.0 -
Vinca 2.3 10.0 4.3 10.0 6.0 10.0
Zinnia 1.0 1.0 1.0 1.0 1.3 1.7
zSour orange, and Viburnum, are hypostomatous with stomates only on the under
surface of their leaves; Pentas and Vinca may be hypostomatous also (not
YTime, seconds for at least 30% infiltration, average for 3 plants.
Table 3. Variation in stomatal infiltration rates of 10 types of
6 plant species.
infiltrating liquids with
Crop Bean Pepper Sour Orange Tomato Viburnum Zinnia
Side of leaf Lower Upper Lower Upper Lower Upper Lower Upper Lower Upper Lower Upper
ET 100 2.5 10.0 7.0 10.0 9.7 -Y 2.0 10.0 7.0 -Y 1.0 1.0
IP 100 2.0 9.0 2.7 8.0 9.3 1.3 5.3 5.3 1.0 1.0
MS 60 MO 40 3.0 4.0 2.0 7.7 3.7 1.3 1.7 6.7 1.0 1.0
SY 2 HO 98 2.0 3.0 3.0 4.0 5.7 2.0 3.3 4.0 2.0 2.0
EP 20 ET 80 1.0 10.0 4.3 6.3 9.3 1.3 10.0 8.0 1.0 1.0
EP 20 IP 80 1.0 10.0 4.0 6.7 9.7 1.3 8.7 5.0 1.0 1.0
EP 20 MS 80 1.0 2.0 1.7 3.0 4.0 1.0 1.0 6.3 1.0 1.0
MA 20 IP 80 1.0 10.0 1.3 2.7 8.0 1.0 2.7 5.3 1.0 1.0
MA 20 ET 80 1.0 10.0 1.7 2.7 9.7 1.3 2.0 5.3 1.0 1.0
MA 50 MS 50 2.0 10.0 1.0 4.7 5.7 1.3 4.0 6.3 1.0 1.0
zET=95% ethyl alcohol
IP=91% isopropyl alcohol
MS= mineral spirits
MO= mineral oil, pharmaceutical grade
SY= Sylgard (organo-silicone wetting agent), Wilbur-Ellis, Fresno,
EP= EOP93 (fatty acids + emulsifiers), Wilbur-Ellis
MA= myristic acid
Y= Readings not made for upper side because leaves only have stomates on under side of
orange and Viburnum.
Time, records for at least 30% infiltration, average for 3 plants.
The Gulf Coast Research and Education Center
The Gulf Coast Research and Education Center is
a unit of the Institute of Food and Agricultural Sci-
ences, University of Florida. The Research Center
originated in the fall of 1925 as the Tomato
Disease Laboratory with the primary objective of
developing control procedures for an epidemic out-
break of nailhead spot of tomato. Research was ex-
panded in subsequent years to include study of sev-
eral other tomato diseases.
In 1937, new research facilities were established
in the town of Manatee, and the Center scope was
enlarged to include horticultural, entomological, and
soil science studies of several vegetable crops. The
ornamental program was a natural addition to the
Center's responsibilities because of the emerging in-
dustry in the area in the early 1940's.
The Center's current location was established in
1965 where a comprehensive research and extension
program on vegetable crops and ornamental plants is
conducted. Three state extension specialists posi-
tions, 16 state research scientists, and two grant
supported scientists from various disciplines of
training participate in all phases of vegetable and
ornamental horticultural programs. This interdisci-
plinary team approach, combining several research
disciplines and a wide range of industry and faculty
contacts, often is more productive than could be ac-
complished with limited investments in independent
The Center's primary mission is to develop new
and expand existing knowledge and technology, and
to disseminate new scientific knowledge in Florida, so
that agriculture remains efficient and economically
The secondary mission of the Center is to assist
the Cooperative Extension Service, IFAS campus
departments, in which Center faculty hold appropri-
ate liaison appointments, and other research centers
in extension, educational training, and cooperative
research programs for the benefit of Florida's pro-
ducers, students, and citizens.
Program areas of emphasis include: (1) genetics,
breeding, and variety development and evaluation;
(2) biological, chemical, and mechanical pest manage-
ment in entomology, plant pathology, nematology,
bacteriology, virology, and weed science; (3) produc-
tion efficiency, culture, management, and counteract-
ing environmental stress; (4) water management and
natural resource protection; (5) post-harvest physiol-
ogy, harvesting, handling and food quality of horti-
cultural crops; (6) technical support and assistance to
the Florida Cooperative Extension Service; and (7)
advancement offundamental knowledge ofdisciplines
represented by faculty and (8) directing graduate
student training and teaching special undergraduate
Q The Institute of Food and Agricultural Sciences,
University of Florida.
D A statewide organization dedicated to teaching,
research and extension.
" Faculty located in Gainesville and at 13 research
and education centers, 67 county extension
offices and four demonstration units throughout
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and renewable resource research and education,
funded by state, federal and local government,
and by gifts and grants from individuals, founda-
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and related sciences and natural resources.
Strengthening Florida's diverse food and
agricultural industry and its environment
Enhancing for all Floridians, the application
of research and knowledge to improve the
quality of life statewide through IFAS exten-