Effect of a Microbial Inoculant on Stomatal Response of Maize Leaves

Abstract

Laboratory tests were conducted to determine the effect of a microbial inoculant on the stomatal response of maize leaves (Zea mays L.). The microbial inoculant investigated is known as Effective Microorganisms or EM and consists of a mixed culture of naturally occurring, beneficial microorganisms. Research has shown that EM applied to soils and plants can improve soil properties and enhance the growth, yield and quality of crops. The exact mechanisms or modes-of-action of how EM cultures elicit beneficial effects on plant growth and metabolism is not known. However, it is likely that some of these cultures can synthesize phytohormones (i.e., auxins and others) or growth regulators that stimulate plant growth. Consequently, the effects of EM and partial illumination on stomatal response of intact and excised maize leaves were evaluated. Potted plants were dried to the wilting point and rehydrated with either a 1:100 dilution of EM and water or water alone applied to the soil. Sudden illumination of plants maintained in the dark showed that the leaf stomata of the EM-treated plants opened more rapidly than water-treated control plants. When leaves were excised and subjected to dehydration, the stomata closed more slowly (i.e., remained open longer) for the EM-treated plants compared with the water-treated control plants. There was no effect of EM on cuticular conductance in any of the experiments. The results of this study indicate that EM cultures contains bioactive substances that can significantly affect leaf stomatal response.

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Effect of a Microbial Inoculant
on Stomatal Response of Maize
Leaves
Hui-Lian Xu a , Xiaoju Wang a & Jihua Wang b
a International Nature Farming Research Center ,
5632 Hata, Nagano, 390-1401, Japan
b Beijing Academy of Agriculture , Beijing, China
Published online: 20 Oct 2008.
To cite this article: Hui-Lian Xu , Xiaoju Wang & Jihua Wang (2001) Effect of
a Microbial Inoculant on Stomatal Response of Maize Leaves, Journal of Crop
Production, 3:1, 235-243, DOI: 10.1300/J144v03n01_19
To link to this article: http://dx.doi.org/10.1300/J144v03n01_19
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Effect of a Microbial Inoculant
on Stomatal Response of Maize Leaves
Hui-lian Xu
Xiaoju Wang
Jihua Wang
SUMMARY. Laboratory tests were conducted to determine the effect
of a microbial inoculant on the stomatal response of maize leaves (Zea
mays L.). The microbial inoculant investigated is known as Effective
Microorganisms or EM and consists of a mixed culture of naturally
occurring, beneficial microorganisms. Research has shown that EM
applied to soils and plants can improve soil properties and enhance the
growth, yield and quality of crops. The exact mechanisms or modes-of-
action of how EM cultures elicit beneficial effects on plant growth and
metabolism is not known. However, it is likely that some of these
cultures can synthesize phytohormones (i.e., auxins and others) or
growth regulators that stimulate plant growth. Consequently, the effects
of EM and partial illumination on stomatal response of intact and ex-
cised maize leaves were evaluated. Potted plants were dried to the
wilting point and rehydrated with either a 1:100 dilution of EM and
water or water alone applied to the soil. Sudden illumination of plants
maintained in the dark showed that the leaf stomata of the EM-treated
plants opened more rapidly than water-treated control plants. When
leaves were excised and subjected to dehydration, the stomata closed
Hui-lian Xu is Senior Crop Scientist and Xiaoju Wang is Research Soil Scientist,
International Nature Farming Research Center, 5632 Hata, Nagano 390-1401, Japan.
Jihua Wang is Crop Scientist, Beijing Academy of Agriculture, Beijing, China.
Address of correspondence to: Hui-lian Xu at the above address (E-mail. huilian@
janis.or.jp).
[Haworth co-indexing entry note]: ‘‘Effect of a Microbial Inoculant on Stomatal Response of Maize
Leaves.’’ Xu,Hui-lian, Xiaoju Wang, and Jihua Wang. Co-published simultaneously in Journal of Crop
Production (Food Products Press, an imprint of The Haworth Press, Inc.) Vol. 3, No. 1 (#5), 2000, pp. 235-243;
and: Nature Farming and Microbial Applications (ed: Hui-lian Xu, James F. Parr, and Hiroshi Umemura)
Food Products Press, an imprint of The Haworth Press, Inc., 2000, pp. 235-243. Single or multiple copiesof
this article are available for a fee from The Haworth Document Delivery Service [1-800-342-9678, 9:00
a.m. - 5:00 p.m. (EST). E-mail address: getinfo@haworthpressinc.com].
E2000 by The Haworth Press, Inc. All rights reserved. 235
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NATURE FARMING AND MICROBIAL APPLICATIONS
236
more slowly (i.e., remained open longer) for the EM-treated plants
compared with the water-treated control plants. There was no effect of
EM on cuticular conductance in any of the experiments. The results of
this study indicate that EM cultures contains bioactive substances that
can significantly affect leaf stomatal response. [Article copies available for
a fee from The Haworth Document Delivery Service: 1-800-342-9678. E-mail
address: getinfo@haworthpressinc.com <Website: http://www.HaworthPress.com>]
KEYWORDS. Cuticular conductance, effective microorganisms, EM,
microbial inoculant, stomatal conductance, Zea mays, excised leaves
INTRODUCTION
There is increasing evidence that mixed cultures of naturally occurring,
beneficial microorganisms applied to soils and plants can improve soil quali-
ty and the growth, yield and quality of crops. One such microbial inoculant is
known as Effective Microorganisms or EM and has been developed by Pro-
fessor Teruo Higa, University of the Ryukyus, Okinawa, Japan (Higa and
Parr, 1994). A number of theories have been proposed as to the modes-of-ac-
tion of EM on plant growth and metabolism (Higa and Wididiana, 1991).
However, the exact mechanisms of how beneficial effects are derived by
either (a) direct effects of microorganisms on the plant or (b) indirect effects
of microbially-synthesized substances (e.g., phytohormones and growth reg-
ulators) are largely unknown. In this regard, Xu et al. (1998) reported that
EM significantly increased the growth and grain yield of maize by promoting
root development and activity that was largely auxin-mediated. Others have
also reported that a number of microbes can synthesize phytohormones and
physiologically-active compounds (Arshad and Frankenberger, 1992; Kam-
pert et al., 1975; Panosyan et al., 1963). However, these reports did not
differentiate between direct effects of microorganisms on plants, or indirect
effects of microbially-synthesized phytohormones. Therefore, the purpose of
this study was to determine whether the liquid stock solution of EM cultures
contained biologically-active substances that could affect stomatal responses
of intact or excised maize leaves to partial illumination and dehydration.
MATERIALS AND METHODS
Plant Materials
Sweet corn plants (Zea mays L. cv. Honey Bantam) were grown in plastic
pots each with a soil surface area of 0.02 m2and a height of 0.25 m. Pots were
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Part II: Microbial Applications 237
filled with a fine textured Andosol. The N-P-K levels in the soil were 3.4,
0.025 and 0.44 g kg−1, respectively, with a C:N ratio of 13. The field
capacity or capillary capacity of the soil was 80% on a gravimetric basis.
Chemical fertilizers used in this experiment were ammonium sulfate (5.3 g
pot−1), long period coated urea (2.8 g pot−1), superphosphate (5 g pot−1)
and potassium sulfate (3.5 g pot−1). One plant was grown in each pot under
glasshouse conditions. The daily temperature and air humidity were not
controlled and fluctuated with ambient conditions.
Pot Treatment
When the 7th leaf was fully expanded, water supply to the pots was
stopped. When the leaves began to wilt, EM was diluted 1:100 with water and
supplied to the pots until the plants were rehydrated (i.e., had regained maxi-
mum turgor).
Partial Illumination
Partial illumination 2000 μmol m−2s−1was applied to the seventh leaf
using an LED light source in the leaf chamber. Other parts of the whole plant
were maintained in dark. Stomatal conductance was measured using a
LI-6400 instrument The stomatal response to partial illumination (Figure 1)
was analyzed using a sigmoid model as follows:
gs=g
max {1/[1 + (1 −βt) e−α(t−τ)]} + gcc(1 + βt),
where, gmax and gcc are the maximum stomatal conductance and cuticular
conductance; βand αare the cuticular and stomatal response constants; t,
time from beginning of illumination; τ, half saturation constant.
Stomatal Closure in Excised Leaves
Stomatal conductance of the 7th leaf of the fully turgid plants was mea-
sured under 2000 μmol m−2s−1until the maximum level was reached. The
leaf was then excised from the plants and measurement continued. The sto-
matal response to leaf dehydration (Figure 2) was analyzed using an expo-
nential model as follows:
gs=[g’
max −gres(1 −β’t)] e−α’(t−τ’) +g
res(1 −β’t)
where g’max is the maximum stomatal conductance before excision; gres is
residual conductance after the stomata are closed; β’and α’are the cuticular
and stomatal response constants; τ’is the time prior to rapid closure of stomata.
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FIGURE 1. A sigmoid model for the response of stomatal conductance (gs)to
partial illumination on the leaves of sweet corn plant in the dark.
0.16
0.12
0.08
0.04
0.00
0 2000 4000 6000 8000
Time (s)
Model
Data
gs(mmol m−2s−1)
FIGURE 2. A model for the decline of stomatal conductance (gs) in an excised
sweet corn leaf blade.
0.20
0.16
0.12
0.08
0.04
0.00
0 2000 4000 6000 8000
Time (s)
Model
Data
gs(mmol m−2s−1)
RESULTS AND DISCUSSION
Stomatal Response to Illumination
Preliminary experiments have shown the response of stomatal conduc-
tance (gs) to partial illumination of sweet corn leaves that had equilibrated in
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Part II: Microbial Applications 239
the dark. The rapid increase in gsupon illumination and the close agreement
between the sigmoid model and experimental data are shown in Figure 1. The
decline in stomatal conductance (gs) that occurs when a leaf is excised from a
corn plant is shown in Figure 2, which indicates close agreement between the
model and experimental data.
When the leaf was partially illuminated in the dark, the stomata started to
open slowly. In this case, the stomata in the EM-treated leaves opened faster
than those of the control plants (Figure 3). The parameters analyzed from the
model are presented in Table 1. The variable gmax shows the maximum gs.
FIGURE 3. Comparison of the sigmoid curves for the response of stomatal
conductance (gs) to partial illumination on the leaf blade of sweet corn plants
treated with EM liquid and water in the dark.
0.18
0 2500 5000 7500
Time (s)
MI
Water
gs(μmol m−2s−1)
0.15
0.12
0.09
0.06
0.03
0
TABLE 1. Parameters of the sigmoid curve of leaf stomatal response to illumi-
nation in the dark for intact leaves of sweet corn plants treated with a microbial
inoculant (EM) compared with water-treated and untreated control plants.
Treatment gmax gcc αβτ
(mmol m−2s−1)(10
−3s−1)(10
−5s−1)(s)
EM-absorbed 0.118 a 0.0398 a −1.10 a 4.01 a 2795 a
Water-absorbed 0.110 a 0.0401 a −1.03 a 2.10 b 2985 b
Control (untreated) 0.113 a 0.0396 a −1.05 a 2.19 b 2953 b
gs, stomatal conductance; gmax,themaximumgs;gcc, cuticular or residual conductance in dark, α,
stomatal response constant; β, residual conductance response constant; τ, time at which stomatal con-
ductance reaches half of the maximum.
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NATURE FARMING AND MICROBIAL APPLICATIONS
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The value of gmax was higher for EM treatment than for water-treated or
untreated plants because EM increased the stomatal opening. The variable gcc
shows the cuticular or residual conductance in the dark. There was no signifi-
cant difference between treatments in this parameter as related to cuticular
characteristics. The coefficient αshows the slope of the fast opening phase of
the stomatal response curve, and βshows the initial slope of the conductance
response curve. The value of βwas higher in the EM-treated plants than in
water-treated or untreated control plants. This means that the EM treatment
increased stomatal opening at the beginning of illumination. Moreover, there
was also a difference in αbetween treatments. The value of αshowed the
stomatal response property and was larger for the EM-treated plants than for
the control plants. The coefficient τshows the time at which stomatal conduc-
tance reaches half of the maximum. The value of τwas higher in the EM-
treated plants than in the water-treated or untreated control plants. This sug-
gests that stomatal conductance was maximized earlier in EM-treated plants
than in the water-treated or untreated control plants.
Decline of Stomatal Conductance in the Excised Leaf Blade
When the leaf was excised from the plant, stomata started to close soon in
response to leaf dehydration. In this case, stomata in the EM-treated leaves
remained open longer than those in the control plants (Figure 4). However,
when stomatal closure reached a fast phase, stomata in the EM-treated leaves
closed faster than those in leaves of control plant.
FIGURE 4. Comparison of the modeling curves for the decline of stomatal
conductance (gs) in excised leaf blades of sweet corn plants treated with EM
liquid and water.
0.20
0.15
0.10
0.05
0.00
0 2000 4000 6000 8000
Time (s)
MI
gs(μmol m−2s−1)
Water
MI-model
Water-model
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Part II: Microbial Applications 241
The parameters analyzed from the model are presented in Table 2. The
variable g’max shows the initial total gsbefore the leaf is excised. As men-
tioned earlier, EM treatment increased stomatal opening and consequently
g’max was higher in EM-treated plants than in water treated or untreated
plants. The variable gres shows the cuticular or residual conductance when
stomata are roughly closed. There was no statistically significant difference
in gres found between these treatments.
The coefficient α’shows the stomatal response constant. The value of α’
was higher for the EM-treated plants than in water-treated or untreated con-
trol plants. The variable gres shows the cuticular or residual conductance.
This indicated that the stomata in leaves of the EM-treated plants closed more
rapidly in response to water loss from the excised leaves compared with the
controls. There was no difference in the residual conductance response
constant (β’) between treatments. Obviously, the short-term treatment with a
microbial inoculant would not change the morphological structure of the leaf
cuticle. The coefficient τ’indicates the time required for rapid stomatal clo-
sure to begin. The value of τ’was higher for the EM-treated plants than for
water-treated or untreated control plants. This suggests that the leaf stomata
in the EM-treated plants could remain open longer under leaf water-deficit
conditions than those of the control plants.
Throughout these experiments, there was no significant effect of EM on
cuticular conductance. Overall, the results indicate that EM contains sub-
stances that can affect stomatal response. Although we do not have direct
evidence to support this conclusion, the various species that comprise EM
used in the present study have been studied extensively for decades. A num-
ber of them are known to synthesize phytohormones, growth regulators and
other biologically-active substances (Arshad and Frankenberger, 1992). Ba-
rea et al. (1976) found that among 50 bacteria isolated from the rhizosphere
TABLE 2. Parameters of the stomatal closure curve for excised leaves of sweet
corn plants treated with a microbial inoculant (EM) compared with water-
treated and untreated control plants.
Treatment g’max gres α’β’τ’
(mmol m−2s−1)(10
−3s−1)(10
−5s−1)(s)
EM-absorbed 0.169 a 0.0505 a −1.24 a 7.4 a 873 a
Water-absorbed 0.158 b 0.0575 a −1.13 b 7.9 a 546 b
Control (Untreated) 0.153 b 0.0593 a −1.16 b 7.1 a 596 b
g’max, the initial total gsbefore the leaf is excised; gres, cuticular or residual conductance when stomata
are roughly closed. α’, stomatal response constant; β’, residual conductance response constant; τ’, time at
which stomatal conductance get into sharp closing course.
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NATURE FARMING AND MICROBIAL APPLICATIONS
242
of various plants, 86, 58, and 90% produce auxins, gibberellins, and kinetin-
like substances, respectively. Kampert et al. (1975) reported that 55% of
bacteria and 86% of fungi isolated from the rhizosphere of Pinus silvestris
could produce gibberellins and their derivatives. Actinomyces and Strepto-
myces produce auxins and similar substances (Purushothaman et al., 1974;
Mahmoud et al., 1984), gibberellins (Arshad and Frankenberger, 1992), and
cytokinins (Bermudez de Castro et al., 1977; Henson and Wheeler, 1977).
Some fungi like Aspergillus niger also produce gibberellins (El-Bahrawy,
1983). The promotion of stomatal responses by intact and excised leaves of
EM-treated plants is likely due to the effects of plant growth regulators
existing in the liquid phase of the cultures. Further studies are needed to
determine the exact mechanisms and modes-of-action whereby these bioac-
tive compounds can affect leaf stomatal responses, growth and metabolism of
plants.
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