Journal of Phytopathology and Pest Management 5(2): 63-87, 2018
pISSN:2356-8577 eISSN: 2356-6507
Journal homepage: http://ppmj.net/
∗
Corresponding author:
R.A.S. Elshafey,
E-mail: relshafey13@yahoo.com
63
Copyright © 2018
Analysis of varietal response to bakanae
infection
Fusarium fujikuroi
and gibberellic acid
through morphological, anatomical and
hormonal changes in three rice varieties
R.A.S. Elshafey
1*
, A.M. Tahoon
2
, F.A. El-Emary
3
1
Rice Research and Training Center, Rice Research Department, Field Crops Research Institute,
Agricultural Research Center, 33717 Sakha, Kafr El-Sheikh, Egypt
2
Rice Pathology Research Department, Plant Pathology Research Institute, Agricultural Research
Center, Giza, Egypt
3
Department of Agricultural Botany, Faculty of Agriculture, Al-Azhar University (Assiut Branch),
71524 Assiut, Egypt
Abstract
Keywords: rice, Gibberella fujikuroi, elongation, bakanae, gibberellic acid, IAA, ABA, anatomical traits.
Fusarium fujikuroi, the causal organism of bakanae disease, is mainly seed borne pathogen
on rice. The response of different rice varieties have more concern to understand
pathogenesis process and host pathogen interaction complex. Therefore, the present study
had some objectives: to determine response of some rice genotypes to bakanae infection
and Gibberellic acid (GA3) treatment through morphological, anatomical and plant
hormones changes. The highly virulent isolate no. 10 of F. Fujikuroi was used in evaluation
of three rice cultivars; Sakha 101, Giza 179 and promising line GZ 10101-5-1-1-1. Changes
in all morphological, anatomical traits and plant hormones activities Gibberellic acid
(GA3), Indol Acetic acid (IAA) and Abscisic Acid (ABA) with bakanae infection and GA3
treatment were assessed from 15-60 days after inoculation and GA3 treatment during
season 2018. Results indicated that bakanae infection caused severe morphological changes
as abnormal elongation, degradation of chlorophyll and seedling death. Morphological
changes were associated with wide anatomical changes of leaf as deformation of motor cell,
mesophyll layer. For stem, infection and GA3 induced significant increase in the No. of
aerenchyma and their diameter and increase pith diameter, and stem elongation. As well
as, anatomical changes in roots were significant increase in diameter of epidermis, cortex
layers, vascular cylinder, and reduction in diameter of xylem vessels. Out of anatomical
results, Fusarium fujikuroi prefer to grow in aerenchyma, pith, cortex, vascular bundle of
both sheath and stem. There is a significant increase in plant hormones Gibberellic acid
(GA3), Indol Acetic acid (IAA) and Abscisic Acid (ABA) with bakanae infection and GA3
treatment combined with bakanae infection and GA3 treatment. GZ 10101-5-1-1-1 was
recorded the lowest response to GA3 treatment with the lowest infection % and stem
elongation%. While Sakha 101 and Giza 179 were the highly susceptible cultivars to
bakanae with the highest infection %, stem elongation% and response to GA3. The fast
and highest stem elongation %, No. of nodes and internode length was considered as
remarkable phenotypic markers it can be used as valuable and early selection marker of
susceptibility in breeding program to bakanae disease. GZ 10101-5-1-1-1 as new promising
line and high tolerant to bakanae and low response to GA3 could be used as a good source
in bakanae resistance breeding program.
Elshafey et al., 2018
64
Introduction
Bakanae caused by the fungal pathogen
Fusarium fujikuroi
Nirenberg
[teleomorph
Gibberella fujikuroi
(Sawada) Ito & Kimura] (Carter et al.,
2008; Leslie & Summerell 2006; Ou,
1985).
Bakanae became more distributed
on large scale with the increase of dry
seeded nursery and direct seeded rice
(Yang et al., 2003). Bakanae
characteristic symptoms are abnormal
elongation of the rice plant and foolish
seedling one of the most important
emerging seed and soil borne disease. In
India, the yield losses ranging from 15-25
and can reached 40 % (Sunder et al.,
2014; Pannu et al., 2012; Rood, 2004).
Highly incidence of bakanae was
demonstrated from Italy (Amatulli et al.,
2012) and almost of major Asian rice
growing countries such as Bangladesh,
Pakistan, India, China, South Korea,
Japan and Taiwan (Chen et al., 2016;
Gupta et al., 2014; Park et al., 2009;
Khan et al., 2000).
Fusarium fujikuroi
,
the causal agent of bakanae disease, was
mainly the most abundant
Fusarium
spp.
(Wulff et al., 2010)
that
isolated from
typical symptoms and only
F. fujikuroi
isolates were able to cause bakanae
disease, (El-Kady et al., 2016; Amatulli et
al., 2010). The fungus have ability to
infect rice plants from pre-emergence to
mature stages, and severe infection of rice
seeds lead to poor germination (Iqbal et
al., 2011).
F. fujikuroi
was
hemibiotrophic fungus, their initial
infection depend on living host cells as a
biotrophic, and progressive infection
includes consumption nutrients and host
cells destruction (necrotrophic) (Ma
et al.,
2013). Gibberellins (GAs) are natural
group of terpenoid plant hormones as
secondary metabolites biomolecules
complex in the fungus
Gibberella
fujikuroi
with strong effects in plant
physiology process. GAs was identified
in some fungal and bacterial species, in
some cases related to virulence
(Cerezo
et al., 2018; Avalos et al., 1999).
Gibberellin producers belong to group C
out of six groups A-F, as subspecies
fujikuroi
the pathogens of rice, with
abnormal elongation as
the most
distinctive symptom of rice seedlings, is
due to the additional growth induced by
the fungal gibberellins
(Klittich & Leslie,
1992; Leslie, 1991). The changes in plant
morphology was contributed to the
ability of
F. fujikuroi
to secrete
gibberellic acids (GAs) as secondary
metabolites (Ou, 1985; Bearder 1983).
The fungus not depend on GAs in growth
and development, as secondary
metabolites although they are thought to
contribute to the virulence of
F. fujikuroi
,
the only Fusarium species capable of
GAs biosynthesis, by controlling
jasmonic acid- responsive gene
expression and jasmonic acid-mediated
plant immune responses (Siciliano et al.
2015; Wiemann et al. 2013). GA
production was also associated with
fungicide sensitivity of different F.
fujikuroi isolates (Tateishi & Suga,
2015). Bioactive gibberellins play an
essential role in many aspects of plant
growth and development, such as stem
elongation, flower and fruit devilment
and seed germination (KO et al., 2006;
Koga et al, 2004; Gomi & Matsuoka,
2003). The ability of fumonisins and
gibberellin GA
3
production in vitro
totally varied with
Fusarium
species.
Whereas fumonisins were produced by
most of the strains of
F. verticillioides
and
F.
proliferatum
, gibberellin GA
3
was
only produced by
F. fujikuroi
(Wulff et
al., 2010). The only
G. fujikuroi
strains
able to produce GAs and the species was
determined as the pathogen of bakanae
disease of rice from total 25 strains
belong 5 Fusarium species (Nur Ain et
al., 2008).
Gibberella fujikuroi
produce
the growth hormone gibberellins, which
Elshafey et al., 2018
65
causes plant elongation (Bhalla et al.,
2010; Berrios et al., 2010).
Plants
produce different types of hormones,
such as gibberellin (GA), auxin, abscisic
acid (ABA), salicylic acid (SA),
ethylene (ET), jasmonic acid (JA),
cytokinin (CK), brassinosteroids (BR)
and strigolactones that may play a role in
plant-pathogen interactions (Bari &
Jones, 2009).
GA and its signaling
components may play important roles in
regulating defense responses against
various necrotrophic and biotrophic
pathogens (Bari & Jones, 2009).
Based
on gibberellin production, 16 strains of
Fusarium moniliforme
and one each
strain of
Fusarium pallidoroseum
,
F.
oxysporium
and
F. solani
observed great
variability in gibberellin production from
different strains of
Fusarium
.
Gibberellins are responsible for the
growth aberrations observed in rice plants
infected with
G. fujikuroi
(bakanae
disease) (Yang et al., 2013). Anatomical
structure of stem was a good indicator to
mechanism of GA
3
application on rice
plant. Therefore, the present study had
some objectives: to determine response of
some rice genotypes to bakanae infection
and GA
3
treatment. In addition to
compare the impact of
F. fujikuroi
and
GA
3
in damage of rice tissues structure
through determine of morphological,
anatomical and plant hormones changes.
Materials and methods
Isolation, pathogenicity and
monitoring changes associated with
Fusarium fujikuroi
infection and
gibberellic acid treatments:
Fusarium
fujikuroi
mainly isolated from infected
upper nodes of stem just above crown of
diseased plants of different rice cultivars.
Small pieces of upper infected nodes and
internodes 1-3 cm were cut and washed
under tap water, sterilized with 3%
NaOCl for 2-3 min, rinsed twice in
sterile distillate water. One-week later
growing mycelia transferred to fresh
PDA media in Petri plates and kept at
room temperature. All isolated Fusarium
isolates were tested in their reaction and
virulence on different rice cultivars. For
proper identification
F. Fujikuroi
, only
isolates of
F. Fujikuroi
that mainly
pathogenic to rice and able to induce
typical symptoms of bakanae disease
(Wulff et al., 2010). The isolated fungus
was identified according to Leslie and
Summerell (2006), Nelson et al. (1983)
and Booth (1971). Preliminary trail was
conducted to determine the proper
concentration of GA
3
whereas five
concentrations at 10, 20, 40, 80 and 160
ppm were added to the nutrients solution.
In addition 15 rice genotypes were used
to select the current tested genotypes and
Minghu 63 was replaced by Giza 179 as
a local cultivar that has the same
response to infection and GA
3
treatment.
Then, the highly virulent isolate no. 10 of
F. Fujikuroi
was used in inoculation of
different rice cultivars. Fungal culture
sub-cultured in PDA at room
temperature. One week later, fungus
mass grown in Petri dishes scraped in
sterile water with a spatula. The final
suspension was filtered through two
layers of sterile cotton lint. Grains of
Sakha 101 the most susceptible cultivar,
Giza 179, Minghu 63 indica japonica and
GZ 10101-5-1-1-1 as highly tolerant
promising line, were soaked in spore
suspension of the tested isolate at
concentration 5
×
10
5
spores / ml for 48h.
Then, all inoculated grains were
incubated for additional two days. All
treatments were arranged as healthy,
infected check and 80 ppm
Elshafey et al., 2018
66
concentration of GA
3
with three
replicates for each cultivars in complete
randomize design. Fifty grains of each
cultivar was seeded in hydroponic
method in nutrient solution in 10 x 10
cm. diameter plastic pots and grown in
the greenhouse at 30-35°C. All
morphological changes as, total shoot
and root lengths measured, plant height,
leaf length, chlorophyll content,
pathological traits combined with
bakanae infection, such as elongation %,
adventitious roots, and bakanae infection
were recorded. The elongation % was
calculated according this formula:
Elongation (%) with GA
3
= (GA
3
treatment –
Control)/Control×100.
Elongation (%) with bakanae infection = (Infected
treatment – Control)/Control×100.
Extraction, separation and estimation
of growth regulating substances:
The
all parts of rice samples were collected
42nd day after sowing, immediately
dipped in liquid nitrogen with 0.5 gm
fresh weigh for each treatment and stored
at frozen conditions. The extraction
method was analyzed at Food
Technology Research Institute,
Agricultural Research Centre according
to Shindy
and Smith (1975) and
Hassanein et al. (2009) as following; to
estimate the amounts of acidic hormones
IAA, ABA and GA
3
, the plant hormone
fractions and standard ones were
methylated according to Vogel (1975) to
be ready for GC analysis. Flame
ionization detector was used for
identification and determination of acidic
hormones using Helwett Packered Gas
Chromatography (5890). The
chromatography was fitted and equipped
with HP-130 mx 0.32 mm x 0.25 mm
capillary column coated with methyl
silicone. The column oven temperature
was programmed at 10°C/min from
200°C (5 min) to 260°C and kept finally
to 10 min. Injector and detector
temperature were 260 and 300°C,
respectively. Gases flow rates were 30,
30, 300 cm/sec for N2, H2 and air,
respectively and flow rate inside column
was adjusted at 2 ml/min. Standards of
IAA, GA
3
and ABA were used. Peak
identification was performed by
comparing the relative retention time of
each peak with those of IAA, GA
3
and
ABA standards. Peak area was measured
by triangulation and the relative
properties of the individual components
were therefore obtained at various
retention times of samples. Elongation
percentage and rice plant hormones
Gibberellic acid (GAs), Indol-3-acetic
acid (I.A.A) and Abscisic acid (A.B.A)
were estimated 30 days after sowing and
the fungus inoculation. The number of
dead seedlings was counted 30 days after
sowing and inoculation.
Anatomical studies:
To investigate the
effect of bakanae disease infection and
GA
3
treatment on some anatomical
changes in structure of leaf, stem, and
roots of rice cultivars;, Sakha 101, Giza
179 and GZ 10101-5-1-1-1. This
investigation was carried out at
laboratory and greenhouse of Rice
Research Center, Sakha, Kafr El-
Sheikh, Egypt and laboratory of
Botany department, Faculty of
agriculture, Assuit, El- Azhar University,
Assiut, Egypt during rice season 2018 .
The samples were taken from all
genotypes after 15 and 60 days after
sowing and seed inoculation of all
treatments to study anatomical structure
of leaf, stem and root by 40 x
Elshafey et al., 2018
67
magnifying. Ten samples of the tip
portion of second leaf, 0.5 cm after
second stem node and 0.5 from the tip of
root were collected of each treatment.
Each sample measured 0.5 cm. All
samples were killed and fixed for 48
hours in FAA (10 ml. formalin, 5 ml
glacial acetic acid, 50 ml ethyl alcohol
and 35 ml water). The dehydrated
samples were infiltrated and embedded in
paraffin (52-54°C m.p.). The embedded
samples were sectioned on a rotary
microtome at a thickness of 5-10 µm.
Sections were mounted on slides and
deparaffinized. Staining was
accomplished with safranine and light
green, cleared in xylol and mounted in
Canada balsam (Gerlach, 1977). Slides
were microscopically examined and
measurements and counts. The sections
were computerized morphometrical
analysis, the morphometrical analysis
was done by Research Microscope type
Axiostar plus made by Zeiss
transmitted light bright field
examinations upgrade able to
professional digital image analysis
system (Carl Zeiss Axiovision Product
Suite DVD 30).
Statistical analysis:
Statistical analysis
conducted using Costat computer
statistical software package, Data were
statistically analyzed according to the
analysis of variance (ANOVA) of the
completely randomized design, applied in
both laboratory and greenhouse
experiments according to Gomez and
Gomez (1984). Least Significant
Difference (LSD) test at 5% was used to
determine genotypic differences among
all means of pathological, morphological
and anatomical traits under each
treatment.
Results and Discussion
Monitoring of pathological and
morphological changes associated with
bakanae infection:
Concerning
morphological changes, invasion of
Fusarium fujikuroi
to rice tissues, fungus
induced significant changes in infected
rice cultivars compared with healthy one.
The earliest symptoms appeared fifteen
days after inoculation, as an inoculated
rice seedling exhibited abnormal
elongation and during this stage, infected
seedling produced early nodes and very
long internodes as characteristic
symptoms to highly sensitive cultivars to
bakanae infection and high response
degree to gibberellic acid (GA
3
)
treatment. Infected seedling became
taller, slender and highly chlorotic than
healthy adjacent seedlings. The
hyperelongation of bakanae infected
plants due to secretion of
F. fujikuroi
to
Gibberellin and plant hormones. The
infected seedlings were thin with
yellowish to pale green leaves (Figure
1a). Tillering ability, the infected
seedlings completely lose their tillering
ability and produce individual plant
during whole season. The healthy
seedling bear two tillers with the main
culm compared with single infected one
(Figure 1b). With longitudinal section of
infected stem (Figure 1b), the internal
surface of upper nodes had dark brown
color and covered with fungal mycelium
mass than white healthy nodes, therefore,
Fusarium fujikuroi
’s recovery percentage
increased from lower nodes to higher-
level nodes, in agreement of Manandhar
(1999). In response of main roots
infection, from lower to upper nodes
production of adventitious roots in
Elshafey et al., 2018
68
progress as the most characteristic
symptoms and specific response to
Fusarium fujikuroi
severe infection
(Figure 1c, d). The infected leaves
became pale yellow and chlorotic with
remarkable degradation of chlorophyll as
chlorosis appearance. In addition, leaves
were severely rolled and converted to
thin slender tubes reflected a wide
reduction in leaf area. Therefore, all
physiological processes will be affected
as a result of chlorophyll damage and
leaf area reduction (Figure 1c, d). The
most evident typical symptoms of
Bakanae are slender thin stem, severe
yellow chlorotic and abnormal elongated
leaves and seedlings that induced as a
result of pathogen production of
gibberellin (Amatulli et al., 2010; Amoah
et al., 1995; Ou, 1987).
Figure 1: Morphological changes combined with infection of F. fujikuroi A: abnormal elongation. B:
tillering ability, C and D: adventitious roots, E: Hyphal growth inside node, F: chlorophyll degradation and
chlorosis., G: leaf rolling, H: healthy, infected and highly infected seedlings.
We selected rice cultivars with divergent
sensitivity to bakanae infection for this
study, Data in Table (1) revealed that
there are significant difference in
response to bakanae infection among rice
varieties. Sakha 101 as highly
susceptible cultivars that exhibited the
highest level of infection percentage
Elshafey et al., 2018
69
58.33 % followed by Giza 179 which
gave 53.33. Whereas, GZ 10101-5-1-1-1
recorded the lowest infection 38.33. Both
cultivars Sakha 101 and Giza 179
reflected high level of susceptible
response to
F. fujikuroi
infection, in
contrary, GZ 10101-5-1-1-1 recorded
high level of tolerance to infection in
comparison to Sakha 101. For elongation
percentage as a good indicator to
response degree to bakanae infection,
elongation percentage was in parallel
with level of infection and highly
associated with bakanae infection and
GA
3
treatment. Data in Table (2) and
Figure (3) indicated that Giza 179 was
superior in their response to infection of
bakanae under artificial inoculation that
gave 50 % over the healthy check. While,
Sakha 101 as a semi dwarf cultivar
showed the second rank of highest
elongation percentage 46.43. Whereas,
GZ 10101-5-1-1-1 as new promising line
have the lowest elongation 20 % taller
over than the check. The increase of GA
3
was found associated with elongation of
internodes and chlorosis of leaves in
susceptible plants of MR 211 (Quazi et
al., 2015). For response to GA
3
treatment, GA
3
treatments were
surpassed infection of bakanae in their
effect of treatment that reflected in
abnormal elongation. Giza 179 was
highly responsive to GA
3
with the
highest elongation percentage, followed
by Sakah 101, while GZ 10101-5-1-1-1
has the lowest response to GA
3
with 27%
compared with 60-62 %. This results in
agreement with Rangaswamy (2012),
and Kwon and Paek (2016) they reported
that gibberellic acid is a plant growth
regulator which produce different effects
comprising stem elongation, enzyme
induction, leaf and fruit senescence,
growth regulation, seed germination and
flowering. The number of nodes and
length of internode 21 days after sowing,
as a good indictor to susceptibility to
bakanae and GA
3
, Sakha 101 and Giza
179 recorded both the highest no. of
nodes and internode length nodes within
3 weeks . out of our results, those both
traits was more associated, fast, more
progressive and specific with infection of
bakanae and susceptibility of cultivar
than GA
3
treatment response. Therefore,
Sakha 101 as highly susceptible cultivar
with high infection developed more
nodes in short time with the highest
length. Then, no. of nodes and length of
internode were good selection traits for
breeding program to bakanae disease as
an early selection marker of
susceptibility.
Table 1: Pathological and morphological changes in rice varieties associated with bakanae infection and GA
3
treatment.
Variety
Treatment
Infection
(%)
Root
length1
5DAI
(cm)
Root
Reduction
(%)
Stem
Elongation
(%)
No. of
nods
Length of
internode
21DAS
(cm)
Dead
plants
21DAS
(%)
Chlorophyll
content
(SPAD)
Leaf
length
(cm)
Droopy
plants %
with
abnormal
elongation
Time after
inoculation
to start drop
(Day)
Sakha
101
Healthy
0.00
13.33
-
0
0.00
0
35.33
6.67
0.00
0.00
Infected
58.33
5.67
57.46
46.43
2.33
6.70
56.67
23.00
25.67
11.67
30.00
80 ppm GA
3
0.00
3.67
72.47
62.50
2
2.33
51.67
21.33
29.67
45.00
25.00
Giza
179
Healthy
0.00
10.31
-
0
0.00
0
32.67
7.33
0.00
0.00
Infected
53.33
7.30
29.19
50.00
1
8.70
53.33
21.33
30.33
91.67
25.00
80 ppm GA
3
0.00
4.33
81.92
60.53
2
6.50
58.33
21.00
35.33
96.00
15.00
GZ101
01-5-1-
1-1
Healthy
0.00
8.35
-
0
0.00
0
34.33
7.33
0.00
0.00
Infected
38.33
6.32
24.31
20.00
0
0.00
11.67
27.67
17.67
1.00
60.00
80 ppm GA
3
0.00
3.33
60.12
27.27
1
3.00
8.33
25.33
23.33
11.67
60.00
L.S.D 5%
2.885
1.013
0.4561
0.645
4.997
1.099
1.033
3.251
1.033
DAI = day after inoculation, DAS = day after sowing.
Elshafey et al., 2018
70
Figure 2: A: Infection with G. fujikuroi and their effect on rice plant elongation
percentage, shoot and root 5 DAI (day after inoculation), B: 15 DAI, C: healthy and
highly infected plants, D: different level of GA
3
.
These results in agreement with Hwang
et al. (2013) revealed that
F. fujikuroi
produced gibberellic acid (GA
3
) which is
involved in plant growth regulation. GAs
accumulate within and around rice roots
during host recognition, pre-penetration
morphogenesis, and pathogen growth in
the plants, and GAs are believed to be
responsible for abnormal internode
elongation of stem because high
concentrations of this hormone cause
hypertrophy of the cells in the parts of
rice found above ground. In extreme
infection, infected plants fall over and
die. The response to GA
3
treatment was
more evident with Giza 179 from
percentage of droopy plants whereas
more than 95 % of tested plants were
started to droop, collapse and lodged
after 15 days from treatment date
compared with GZ 10101-5-1-1-1 that
gave almost 12 % lodging after 60 days
as very late response (Table 1 and Figure
3). These results in harmony with Quazi
et al. (2015) reported that with severe
symptoms progression of bakanae
infection and increase of GA
3
, after 21
days of inoculation, susceptible plants of
MR 211 were started to collapse and
lodged due to over elongation.
Elshafey et al., 2018
71
Figure 3: Effect of infection of G. fujikuroi and GA
3
treatment 80 ppm on rice plant
elongation percentage and droopy plants of rice cultivars Sakha 101, Giza 179 and GZ
10101-5-1-1-1.
The damage and deleterious effect of
both bakanae infection and GA
3
was
clear in reduction of root length that
ranged with bakanae infection from
24.31 to 57.46 and GA
3
treatment caused
remarkable reduction ranged from 60.12
to 81.92 % (Table 1, Figure 1a, Figure 2
a, b and d). In addition, the chlorophyll
content of GZ 10101-5-1-1-1 more stable
for long time and their content not
affected more than infected plants,
whereas healthy plants have 34.33 SPAD
and infected plants 27.67 and GA
3
treatment 25.33 with low significant
differences. The low degradation of
chlorophyll and chlorosis was a good
indicator for tolerance of GZ 10101-5-1-
1-1 to bakanae infection. These results
are in agreement with Hwang et al.
(2013) who reported that isolate FfB20
of
F. fujikuroi
triggered rice seedling
elongation and foolish growth. In
addition, symptoms on rice roots
inoculated with FfB14 or FfB20 isolates,
drastically inhibited root growth, and the
roots and crowns were decayed. In trial
on various concentrations and effect of
GA
3
on Minghu 63 with 10, 20, 40, 80
and 160 ppm, GA
3
exhibited the same
behavior and effect of
F. fujikuroi
that
induced the reduction of root length and
induction of abnormal elongation with
increase of GA
3
concentration (Figure
3d). GA
3
concentration at 80 ppm
recorded the same effect of 160 ppm, so
we used 80 ppm in study of anatomical
changes. Gibberellins are responsible for
the growth aberrations observed in rice
Elshafey et al., 2018
72
plants infected with
G. fujikuroi
(Yang et
al., 2013). According to pathological and
morphological response to bakanae
infection and GA
3
, GZ 10101-5-1-1-1
exhibited more desirable traits for
bakanae tolerance as late response to
GA
3
, low infection of bakanae, more
stable chlorophyll content. Finally, we
can recommend, according our results,
using this line as a high tolerance
resource in a successful bakanae disease
breeding program.
Table 2: Anatomical changes in leaf of rice cultivars associated with bakanae infection and GA
3
treatment.
Variety
Treatment
Leaf
thickness
(μ)
Mesophyll
(μ)
Upper
epidermis
(μ)
Motor cell
length
(μ)
Motor cell
width
(μ)
Bundle
sheath (μ)
Bundle Ø
(μ)
M.xylem Ø
(μ)
Midrib
length
(μ)
Midrib
width
(μ)
Sakha 101
Healthy
95.64
70.98
14.25
53.54
81.8
27.22
102.06
31.78
169.00
147.89
Infected
65.46
31.95
24.64
25.70
42.1
22.74
114.78
22.74
140.54
114.45
80 ppm GA
3
69.26
36.25
21.59
26.66
41.5
29.70
101.35
19.75
135.65
139.65
Giza 179
Healthy
102.86
71.24
18.48
52.43
74.2
39.27
176.47
27.19
170.38
160.87
Infected
82.77
52.63
20.61
24.30
50.1
26.35
115.93
16.9
110.56
128.76
80 ppm GA
3
71.57
45.54
15.17
26.47
37.6
16.40
93.85
14.03
125.78
131.95
GZ101 01-
5-1-1-1
Healthy
119.24
95.00
16.65
51.51
80.6
50.65
118.9
25.64
172.89
170.34
Infected
105.32
79.93
13.51
48.57
72.7
44.19
120.72
22.12
166.15
164.81
80 ppm GA
3
100.67
81.99
14.15
48.91
67.4
38.59
111.49
17.2
156.79
156.37
L.S.D 5%
4.433
5.933
3.642
3.723
11.28
3.587
4.537
3.328
6.264
4.632
Leaf anatomical changes associated
with bakanae infection and GA
3
treatment:
Concerning the effect of
Bakanae disease infection on leaf
anatomy, F. fujikuroi infection induced a
significant change in the anatomical
structure of the third leaf of rice
seedlings at 21 DAS (Figure 4 and Table
2). Data revealed that some tissues were
decreased, while the others increased in
thickness or diameter as a clear response
to the fungus infection. The third leaf
thickness, mesophyll, bundle sheath
thickness and midrib width, in the
infected plants were reflected highly
significant reduction in opposite to
healthy plants. For leaf damage, some
anatomical changes reflected
morphological damage due to infection
such as reduction in leaf area and rolling.
Figure 4a and b illustrated that the
fungus infection caused a sever
reduction in leaf area and rolling
compared with full stretched healthy
leaves (Figure 4c). Regarding leaf
thickness, both susceptible cultivar
Sakha 101 and Giza 179 recorded highly
significant reduction compared with
tolerant one GZ 10101-5-1-1-1.
Whereas, thickness of infected Sakha
101 leaves was 65.46 μ compared to
healthy 95.64 μ on the other hand GZ
10101-5-1-1-1 was 105.32 μ opposite to
119.24 μ, (Table 2 and Figure 4F, G).
Therefore, tolerance of GZ 10101-5-1-1-
1 was clear evident from many traits
such as, stable leaf thickness. The same
trend of reduction was recorded with
different tissues; Mesophyll layer,
bundle sheath thickness and midrib.
Elshafey et al., 2018
73
Figure 4: Transverse section of rice leaf and anatomimcal structure due to G, fujikuroi
Infection. A: infected rice leaf 15 DAI. B: Rolling of infected leaf 15 DAI. C: Health leaf. D
and E: health and infected Midrib region. F, G and H: health, infected and GA
3
treatment. I,
M and N: health, infected and GA
3
paranchyma cell of midrib. J, K and L: health, infected,
GA
3
motor cell and epidermal cell. O and P: health and infected midrib. Q, R and S: VB of
midrib. Bar=50µ.
For mesophyll layer thickness, it was
sharply decreased by more than two
times compared with the healthy leaves,
while, infected plants of Sakha 101 gave
31.95 compared to 70.98 μ. On the other
hand, GZ 10101-5-1-1-1 exhibited low
reduction in Mesophyll layer thickness.
The reduction in a Mesophyll thickness
was highly associated with sharp
reduction in chlorophyll content and
chlorosis, so it should significantly affect
photosynthesis process and subsequently
reduces grain yield of infected plants.
Therefore, this explained that GZ 10101-
5-1-1-1 still have high chlorophyll
content after bakanae infection compared
with both Sakha 101 and Giza 179. For
GA
3
treatment, GA
3
exhibited the same
trend and impact of reduction on all
aforementioned traits. Subsequently,
GA
3
have the same behavior of
F.
fujikuroi
infection in anatomical changes
of leaf. For motor cells, both dimensions
of motor cell was sharply decreased with
bakanae infection and GA
3
treatment,
more than 50 % in both Sakha 101 and
Giza 179, in the opposite direction motor
cell of GZ 10101-5-1-1-1 not affected.
So, the leaf rolling and deformation as a
result of bakanae infection and GA
3
treatment was highly associated with
damage of motor cells, subsequently the
lamina be rolled (Table 2 and Figure 4B,
J, K, L). Out of our results, all leaf
anatomical changes with infection and
GA
3
treatment was highly associated
Elshafey et al., 2018
74
with and morphological changes, and
reflected damage in all leaf tissues. The
response of xylem and vessel diameter to
infection was clear in reduction with
almost 40-50 % of Sakha 101 and Giza
179 with infection and GA
3
treatment.
The damage of xylem vessel was sharply
and negatively affected water absorption,
water content, nutrients and starch
translocations. Also, the rest of
anatomical traits such as bundle sheath μ,
bundle Ø, and midrib dimensions μ were
decreased in response to infection and
GA
3
treatment (Table 2 and Figure 4, F-
H, I, M, N, O-S). Only upper epidermis
layer exhibited an increase in its cells
diameter response to infection and GA
3
opposite behavior to all other anatomical
leaf traits (Table 2 and Figure 4, F-H).
So, deformation of leaf structure due to
bakanae infection and GA
3
treatment, all
growth process and nutrients
translocation will be affected. The
deformation of leaf structure actually
will be associated with yield loss.
Anatomical structure of flag leaf of
susceptible rice cultivars significantly
changed in response to bakanae infection
and GA
3
treatments. All morphological
and anatomical changes in leaf structure
of rice were matched with the same
changes that caused by white tip
nematode infection. These findings are in
accordance with
Artyukhova and Popova
(1981) and Elshafey et al. (2010) who
recorded ultrastructural changes caused
by
A. besseyi
to leaves of susceptible rice
plants. They reported that nematode
causes crimping and chlorosis,
considerable changes in the lamina, the
structure of the epidermis, and
misalignment, underdevelopment and
deformation of the motor cells. Also,
these results are in harmony with
findings of Jairajpuri and Baqri (1991),
who reported that the injury due to the
infection of
A. besseyi
caused by the
stylet leads to the disintegration of
phloem cells. It is clear that white tip
nematode disease deforms and damages
the anatomical structure of rice flag leaf,
which is the important organ for
photosynthesis.
Aphelenchoides besseyi
caused a sharply decrease in the
chlorophyll content of the cell, so the
photosynthesis rate is severely and
negatively affected. According to the
deformation of flag leaf due to infection
of white tip nematode all agronomic
traits will be decreased. White tip
nematode infection deformed the motor
cells which controlled the rolling and
expanding of leaf blade, so the leaf area
which exposed to the light of sun sharply
diminished as a result of this damage the
photosynthesis rate reached to the lower
level. Vascular bundle, xylem diameter
were negatively affected with white tip
nematode infection as a result of this
damage the translocation of nutrients
from source to the sink will be affected.
As a result of all above-mentioned
damages, the growth of the plant will be
affected, these findings in accordance
with results of Tahoon (2016) reported
that bakanae infection caused a reduction
and loss in chlorophyll content, leaf area,
abnormal elongation and produce small
panicles with high sterility. The bakanae
infection and GA
3
decreased most of leaf
anatomical characters. The tolerant and
susceptible genotypes revealed wide
variation and differentiating parameters
in leaf anatomy.
Elshafey et al., 2018
75
Sheath anatomical changes associated
with bakanae infection and GA
3
treatment:
Bakanae disease infection
induced significant changes in the
anatomical structure of sheath of rice
cultivars. Figure (5) illustrated that
tissues of highly infected cultivar Sakha
101 reached to senescence after 21 days
from inoculation, this was clear in Figure
(5a) with sheath of health plants
compared with infected ones in Figure
(5B, C), red arrows in Figure (5C) refers
to accumulation of unknown substances
in infected cells. In addition, air space or
Aerenchyma of infected sheath (Figure
5D) was full occupied with
Fusarium
hyphal mass compared with health
tissues (Figure 5H). Whereas, the health
tissues of sheath can reached the
senescence conditions within 60 days
after sowing (Figure 5F, G).Therefore,
infection of
Fusarium fujikuroi
induced
senescence of infected tissues in short
period of time than healthy leaves and
blocking of aerenchyma with growth of
fungus reduced the amount of available
air and respiration process. The growth
of fungus inside tissues affected all
growth process and consume nutrients.
Consequently, the yield was negatively
affected with the infection of bakanae
disease. The fungus significantly
induced abnormal elongation of sheath
cells (Figure 5K) compared with health
cells Figure (5I, J). These findings are in
agreement with Hwang et al. (2013)
found that
F. fujikouri
vigorously
infected rice stems.
Figure 5: Transverse section of rice sheath and anatomical structure due to F. fujikuroi
Infection. A: health rice leaf inside sheath 21 DAI. B: infected sheath and senescence 21
DAI, sheath. C: accumulation of unknown substances inside infected cells 21 DAI. D: hyphal
growth in Aerenchyma (ARC), H: healthy and free ARC. E: Healthy sheath. F, G:
accumulation of unknown substances inside healthy cells 60 DAS. I: longitudinal section of
health sheath. J: infected leaf. K: GA
3
treated sheath, bar 100µ.
Elshafey et al., 2018
76
Stem anatomical changes associated
with bakanae infection and GA
3
treatment:
Data presented in Table (3)
and Figure 6 showed that, bakanae
infection and GA
3
treatment, in the
present study increased the most varieties
stem anatomical features compared with
health plants. The significant increase
was evident in the no. of aerenchyma and
their diameter. Whereas, the number of
aerenchyma increased from 0 in health
plants to 14 in highly susceptible of
Sakha 101 and 7 aerenchyma with Giza
179, while only one air space
(aerenchyma) was formed in infected
stem of tolerant variety GZ 10101-5-1-1-
1. In addition, there is significant
increment in diameter of this aerenchyma
with infection and GA
3
treatment.
Aerenchyma (Gas spaces) forms as an
adaptation to submergence to facilitate
gas exchange. In rice (
Oryza sativa
),
aerenchyma develop by cell death and
Lysis, and H
2
O
2
promotes aerenchyma
formation in a dose-dependent manner
(Steffens et al., 2010). Cell death in
response to biotic or abiotic stresses is
often mediated by plant hormones. In
addition, reactive oxygen species (ROS)
superoxide anion radical (O
2
-
) and
hydrogen peroxide (H
2
O
2
) are central
regulators of plant cell death (Bouchez et
al., 2007; Overmyer et al., 2003; Moeder
et al., 2002).
The infection of
Fusarium
and GA
3
treatment may be induced Lysis
and cell death of stem cell, free radicals
release such as O
2
-
and H
2
O
2
therefore,
increase the aerenchyma formation. This
fungus was aerobic and prefers aeration
for production of GA
3
and this aeration
condition surely necessary because the
route of the biosynthesis of GAs
involves a series of oxidative steps.
Therefore, the microorganism’s demand
for oxygen may increase with the growth
of mycelium. Oxidative steps, which are
catalyzed by citocromo P450
monooxygenases, dioxygenases and
dehydrogenases, a high aeration
condition is critical for an optimal
production process (Tudzynski, 2005;
Machado et al. 2001; Tudzynski, 1999).
Therefore, for more aerobic conditions
F. fujikuroi
demand wide aerenchyma
number and were available in highly
susceptible cultivars than resistant.
Table 3: Anatomical changes in stem tissues of rice cultivars associated with bakanae infection and GA
3
treatment.
Variety
Treatment
No. of
aerenchyma
Aerenchyma
*
Ø (μ)
Vascular
bundle
Ø (μ)
No. of
Vascular
bundle
M.
xylem
Ø (μ)
Pith
Ø (μ)
Ground
tissues
Ø (μ)
Stem
Ø (μ)
Stem cell
length
**
(μ)
Stem cell
elongation
(%)
Sakha 101
Healthy
0.00
0.0
139.8
29.33
23.0
110.20
41.40
1649.30
23.72
-
Infected
14.33
186.0
142.8
29.67
30.0
634.60
66.50
1660.00
44.20
46.33
GA
3
80 ppm
9.33
223.0
233.8
11.00
69.0
271.50
97.10
2008.30
58.96
59.77
Giza 179
Healthy
0.00
0.0
141.8
29.67
22.0
107.60
38.40
1641.70
17.97
-
Infected
7.33
187.0
161.8
13.67
40.0
428.50
65.40
1216.30
38.56
53.40
GA
3
80 ppm
7.33
197.0
194.4
13.67
66.0
518.80
124.10
1683.80
46.09
61.01
GZ 10101-
5-1-1-1
Healthy
0.00
0.0
145.1
30.00
23.0
109.80
38.70
1655.10
16.36
-
Infected
1.00
108.0
299.8
12.67
66.0
417.70
89.10
2338.80
30.21
45.85
GA
3
80 ppm
1.00
158.0
384.7
12.00
116.0
461.50
185.80
2838.70
31.03
47.28
L.S.D 5%
0.807
100.7
28.49
0.950
7.5
22.20
12.58
21.21
6.634
*
diameter of average no. of aerenchyma,
**
longitudinal section of stem.
Elshafey et al., 2018
77
Figure 6: Transverse section of rice stem and anatomical structure due to F. fujikuroi
infection and GA
3
80 ppm treatment. A: health stem of Sakha 101. B: infected stem of
Sakha 101. C: GA
3
treated stem of Sakha 101. D, E, F: health, infected and GA
3
treatment of Giza 179. G, H, I: health, infected and GA
3
treatment of GZ10101-5-1-1-
1, bar 200µ. VB: Vascular bundle.
The anatomical features that increased
with infection and GA
3
treatment were
cleared in Vascular bundle Ø, M. xylem
Ø, Pith Ø μ, Ground tissues Ø, and Stem
cell elongation %. The invasion of
Fusarium fujikuroi
to vascular bundle
and surrounded tissues of GT induced a
significant increase in diameter of M.
xylem and this may be contributed to
feeding of this fungus on cell wall and
secretion of different cell lytic enzymes
such as cellulose increase weakness and
breaking down of cell wall. Therefore, it
caused easy expanded and stretched of
cell wall of infected cells and
consequently increased in diameter
(Table 3 and Figure 7). our results
illustrated that the
Fusarium
fungus have
a wide preference in their growth to
occupy pith area and progress in growth
through the direction to GT cells and
finally vascular bundles (Figure 7B-F).
Our results in line with Hwang et al.
(2013) who reported that Invasive
mycelial growth of isolates FfB14 and
FfB20 in the rice stem harvested at 7
dpi. FfB14 vigorously infected rice
stems, and active invasive mycelial
growth was observed. FfB20 also grew
in the rice stem; however, the growth
was relatively lower. In addition
revealed the active fungal growth of
FfB14 in the root and crown of rice
seedlings, while the growth rate of
FfB20 in rice was more than 4 times
lower than FfB14. Massive infective
Elshafey et al., 2018
78
mycelial growth of FfB14 was evident in
rice stems and crown; however, FfB20
did not exhibit vigorous growth. The role
of GA
3
was more marked and evident in
stem cell elongation % and it was clear
that this fungus have the ability to induce
production of GA
3
, therefore infection of
Fusarium exhibited almost the same level
of elongation of internodes. In addition,
the same trend of cell response with
infection of
Fusarium
to all stem tissues
and effect of GA
3
indicated that GA
3
paly an essential role in infection process
and both virulence and susceptibility of
cultivars. The tested varieties exhibited a
wide variation in their response to both
infection and GA
3
. The tolerant variety
GZ 10101-5-1-1-1 reflected various
responses than highly susceptible
cultivar Sakha 101, whereas, GZ 10101-
5-1-1-1 recorded the highest increment
in all stem anatomical features specially
Vascular bundle Ø, M. xylem Ø, Ground
tissues, low stem elongation % and this
increment in side of bakanae tolerance
(Table 3 and Figure 6).
Figure 7: Transverse section of rice stem and anatomical structure due to G. fujikuroi
infection. A: health stem. B, C, D: invasion and development of infection in vascular
bundle. E, F: growth of Fusarium fujikuroi in pith and cortex cells. G: longitudinal sections
of health stem cell. H: longitudinal sections of infected and abnormal elongated cell, bar
100µ. VB: Vascular bundle, ph: phloem.
Elshafey et al., 2018
79
GZ 10101-5-1-1-1 was recorded the
lowest response to GA
3
treatment,
whereas the sensitivity to GA
3
combined
with highest stem elongation % as with
Giza 179 as 60 % compared with 47.28
% of GZ 10101-5-1-1-1. The high
sensitivity to GA
3
associated with high
level of bakanae susceptibility. The fast
and highest stem elongation % was
considered a remarkable phenotypic
marker it can be used as valuable
selection marker in breeding program to
bakanae disease.
Root anatomical changes associated
with bakanae infection and GA
3
treatment:
Data presented in Table (4)
and Figure (8) showed that, bakanae
infection and GA
3
treatment, in the
present study increased the most varieties
root anatomical features compared with
health plants except M. xylem vessels Ø.
The increment in diameter or cell
expansion have a fixed direction from
epidermal cell to cortex until reached to
vascular cylinder. Therefore, the
hypertrophy response of for-mentioned
root tissues caused a stress and pressure
on M. xylem vessels; consequently, the
diameter of M. xylem will be reduced
and blocked. The reduction in M. xylem
diameter will be associated with deficit
of water absorption resulted in low water
content and wilt.
Table 4: Anatomical changes in root tissues of rice cultivars associated with bakanae infection and GA
3
treatment.
Variety
Treatment
Epidermal
thickness (μ)
Cortex
thickness (μ)
Cortex cell
diameter Ø (μ)
Vascular cylinder
Ø (μ)
M. xylem vessels
Ø (μ)
Sakha 101
Healthy
28.84
138.69
18.79
111.27
15.87
Infected
60.60
159.74
42.29
136.02
7.51
GA
3
80 ppm
58.00
165.03
47.10
164.80
7.14
Giza 179
Healthy
30.97
134.50
17.53
142.95
11.25
Infected
54.70
150.70
39.44
169.12
7.56
GA
3
80 ppm
53.66
158.73
43.80
178.89
7.01
GZ 10101-5-1-1-1
Healthy
29.33
137.48
17.15
146.85
11.43
Infected
44.90
145.64
32.18
158.24
8.39
GA
3
80 ppm
51.78
151.91
41.24
168.22
7.24
L.S.D 5%
4.971
6.031
4.120
4.302
2.295
The infection of
Fusarium fujikuroi
to
root tissues caused collapse, breakage of
cell wall, degradation and finally cell
Lysis and death as clear in Figure (8F).
Finally, Sakha 101 and Giza 179
exhibited the highest response in all root
anatomical traits compared with GZ
10101-5-1-1-1 and healthy check. Data in
Table (5) represented the correlation
among infection of bakanae disease, GA
3
treatment, morphological, anatomical,
plant hormones traits of rice verities.
Data indicated that there a highly
significant and positively correlation
among infection of bakanae infection and
some anatomical features such as
elongation percentage, number of
aerenchyma, aerenchyma Ø, stem cell
length, stem cell elongation, epidermal
thickness, cortex thickness (μ), cortex
cell diameter Ø, stem vascular cylinder Ø
(μ), stem M. xylem vessels Ø, GA
3
.
Whereas, highly significant and
negatively correlated with chlorophyll
Elshafey et al., 2018
80
content, leaf thickness (μ), motor cell
length (μ), motor cell width (μ), leaf
bundle sheath (μ), bundle Ø, leaf M.
xylem Ø, midrib length (μ), number of
stem vascular bundle, stem M. xylem
vessels Ø (μ), root M. xylem vessels Ø.
Table 5: Correlation coefficients among infection of F. fujikuroi, GA
3
treatment and some morphological,
anatomical, plant hormones traits of rice verities.
Infection
Elongation
Chlorophyll
GA3
IAA
ABA
Infection (%)
1
0.966
**
-0.981
**
0.743*
0.669*
0.327
Elongation (%)
0.966
**
1
-0.970
**
0.808**
0.540
0.341
Chlorophyll content
-0.981
**
-0.970
**
1
-0.745*
-0.637
-0.416
Leaf length (cm)
0.975
**
0.975
**
-0.983
**
0.838**
0.539
0.384
Leaf thickness (μ)
-0.805
**
-0.865
**
0.788
*
-0.659
-0.504
-0.214
Mesophyll (μ)
-0.746
*
-0.821
**
0.741
*
-0.586
-0.524
-0.238
Upper epidermis (μ)
0.383
0.471
-0.437
0.057
0.534
0.350
Motor cell length (μ)
-0.878
**
-0.940
**
0.894
**
-0.731*
-0.598
-0.492
Motor cell width (μ)
-0.918
**
-0.965
**
0.925
**
-0.789*
-0.530
-0.282
bundle sheath (μ)
-0.622
-0.687
*
0.602
-0.711*
-0.279
-0.247
bundle Ø
-0.530
-0.536
0.428
-0.521
-0.125
-0.050
M.xylem Ø
-0.842
**
-0.809
**
0.879
**
-0.777*
-0.384
-0.352
midrib length (μ)
-0.829
**
-0.893
**
0.881
**
-0.783*
-0.505
-0.675*
midrib width (μ)
-0.713
*
-0.752
*
0.704
*
-0.541
-0.589
-0.427
No. of aerenchyma
0.798
**
0.829
**
-0.780
*
0.496
0.686*
0.306
Aerenchyma Ø
0.984
**
0.967
**
-0.985
**
0.697*
0.635
0.346
Vascular bundle
0.310
0.162
-0.292
0.045
0.132
-0.170
No. of Vascular bundle
-0.693
*
-0.650
0.710
*
-0.586
-0.286
-0.265
M. xylem Ø
0.509
0.409
-0.505
0.330
0.116
-0.122
Pith Ø
0.853
**
0.715
*
-0.803
**
0.494
0.780*
0.296
Ground tissues (μ)
0.559
0.461
-0.552
0.423
0.117
-0.127
Stem Ø
0.098
-0.052
-0.051
-0.173
-0.009
-0.477
Stem cell length (μ)
0.903
**
0.951
**
-0.883
**
0.692*
0.498
0.172
Stem cell elongation
0.977
**
0.925
**
-0.964
**
0.709*
0.638
0.330
Epidermal thickness (μ)
0.970
**
0.929
**
-0.966
**
0.592
0.735*
0.350
Cortex thickness (μ)
0.938
**
0.947
**
-0.909
**
0.666
0.538
0.125
Cortex cell diameter Ø (μ)
0.973
**
0.944
**
-0.965
**
0.677*
0.602
0.257
Vascular cylinder Ø (μ)
0.671
*
0.654
-0.732
*
0.686*
0.213
0.272
M.Xylem vessels Ø (μ)
-0.872
**
-0.808
**
0.900
**
-0.565
-0.619
-0.280
GA
3
0.743
*
0.808
**
-0.745
*
1
0.102
0.195
IAA
0.669
*
0.540
-0.637
0.102
1
0.521
ABA
0.327
0.341
-0.416
0.195
0.521
1
Gibberellic acid activity changes with
bakanae infection and GA
3
treatment:
Data presented in Table (5) indicated
that, there was a remarkable increase in
GA
3
(ppm) associate with infection of
bakanae and GA
3
treatment in
comparison with healthy plants. For
correlation coefficients, GA
3
was gave
highly significant and positively
correlation infection (%), infection (%),
elongation (%), leaf length (cm), upper
epidermis (μ), aerenchyma Ø, stem cell
length (μ), stem cell elongation, cortex
cell diameter Ø (μ), root vascular
Elshafey et al., 2018
81
cylinder Ø, while was highly significant
and negatively correlated with
chlorophyll content, motor cell length
(μ), motor cell width (μ), bundle sheath
(μ), M.xylem Ø, midrib length (μ),
midrib length (μ) (Table 5).
Figure 8: Transverse section of rice root and anatomical structure due to G. fujikuroi
infection. A: health root of Sakha 101, 21 DAI. B: infected with bakanae. C: treated with
80 ppm GA
3
. D: health section 60 DAI. E: infected section. F: highly damaged roots.
Bar= 100 µ, Ep, epidermal cells. Co: Cortex. V.C.: vascular cylinder. X.V: xylem
vessels. ARC: Aerenchyma. M.xy: Meta xylem.
Indol acetic acid activity changes with
bakanae infection and GA
3
treatment:
For the changes in activity of indol acetic
acid activity (IAA), data presented in
Table (5) indicated that, there was
increasing in IAA (ppm) under all
infection percentage compared with
healthy plants in all rice cultivars under
the present study. For correlation
coefficients, IAA was gave highly
significant and positively correlation
infection (%), elongation (%), leaf length
(cm), upper epidermis (μ), aerenchyma
Ø, stem cell length (μ), stem cell
elongation, cortex cell diameter Ø (μ),
root vascular cylinder Ø, while was
highly significant and negatively
correlated with chlorophyll content,
motor cell length (μ), motor cell width
(μ), bundle sheath (μ), M. xylem Ø,
midrib length (μ), midrib length (μ)
(Table 5).
Elshafey et al., 2018
82
Table 6: Plant hormones changes through rice cultivars associated with bakanae infection and GA
3
treatment.
Treatment
Plant hormones in rice plants (ppm)
Sakha 101
Giza179
GZ 10101-5-1-1-1
GA
IAA
ABA
GA
IAA
ABA
GA
IAA
ABA
Healthy plants
350
0.8
0.7
310
2.5
0.5
300
0.7
0.7
Infected plants
500
20
2.3
850
15
10.5
420
15
1.7
GA3 (80 ppm)
850
8.2
0.8
1520
5
0.8
510
6.5
0.9
Abscisic acid activity changes with
bakanae infection and GA
3
treatment:
Concerning the abscisic acid (ABA)
(ppm) data in Table (5) cleared that there
was positively impact on ABA (ppm)
with increase infection of
F. fujikuroi
in
the present study, were its led to increase
it compared with healthy plants in the
tree cultivars under the present study.
Abscisic acid probably compromises rice
defense against pathogens. Jiang et al.
(2010) demonstrated that ABA
suppresses the basal resistance of rice
when it interacts with the pathogen
Pyricularia oryzae. For the situation and
relationship among different hormones,
Elemary et al. (2015)
demonstrated that
abscisic acid (ABA) was higher in the
untreated entries or varieties with GA
3
.
The highest value of ABA was observed
with untreated IR69625A. For Indol
acetic acid (IAA) showed highly
significant and positive correlation with
plant height, amylose content% and GA
3
,
While, abscisic acid showed significant
and negative correlation with plant
height, number of panicles per plant,
seed set %, grain yield per plant all
results in agreement with Kim et al
(2014) reported that, ABA able to
improve abiotic stress tolerance in rice,
fine regulation of its expression will be
required to avoid deleterious effects on
agricultural traits. In addition
,
Quazi et
al. (2015) found that the increase in GAs
including fungi produced GA3 and IAA
were higher in inoculated susceptible
MR 211 rice plants (GAs = 26%, IAA =
40.39%) as compared to resistant BR3
(GAs = 19%, IAA = 4.27%), 7 days after
inoculation. 14 days after inoculation
both phytohormones were observed to
increase., after 21 days of inoculation
with abnormal elongation, and finally
dead followed by a decrease in GAs and
IAA but an increase in ABA. In resistant
variety BR3, marginal up regulation of
GAs and IAA were observed only at 21
days after inoculation in stems with no
typical symptom of bakanae disease. The
increase of infection combined with
remarkable increase of GA levels with
both cultivars Sakha 101, Giza 179 and
GZ 10101-5-1-1-1. In addition, GA3
increase with infection of bakanae
disease and treatment of GA3. Infection
of bakanae induced increase of IAA and
ABA. While, GA
3
application was more
associated with increase of IAA than
ABA (Table 6). All results in agreement
with Tahoon (2016) who reported that
correlation coefficient values indicated
that there are significantly and positively
correlation among infection % and
elongation %, GA, IAA and ABA. Also,
elongation percentage was positively
correlated with concentration of GA and
both IAA and ABA. GA content
positively correlated with both IAA and
ABA in both Sakha 101 and Giza 179
cultivars. Considering the synergistic
relationship between GA
3
and IAA, and
Elshafey et al., 2018
83
antagonistic relationship between GA3
and ABA (Chen et al., 2006; Xu et al.,
1998), it was postulated that GA 3, IAA
and ABA phytohormones might be
involved in different bakanae symptoms
expression. As endogenous GA 3 levels
were found to be correlated with
elongation (hypertrophy) in bakanae
diseased plants (Kuo & Yang. 1967),
therefore, the justification of the current
study was that the up-regulation level of
GA
3
might also be responsible for the up-
and down- regulation of IAA and ABA
and thereby associated with other
symptoms expression. Moreover, in
relation to bakanae disease development,
IAA and ABA that might have influences
on disease susceptibility/ resistance and
have not been identified. Conversely, the
concentration of abscisic acid remained
constant in the resistant cultivar;
however, the levels of this phytohormone
were highest in the inoculated plants
compared to the uninoculated control
(Bolwell et al., 2002; Blee et al., 2001).
Accumulation of ethylene and GA and a
decreased ABA level in the rice
internode thus favor induction of
epidermal cell death and ensure that
Programmed cell death (PCD) is initiated
as an early response that precedes
adventitious root growth. PCD further
promoted by gibberellin (GA).
Gibberellic acid was also shown to
promote ethylene-induced cell death,
while abscisic acid (ABA) acts as a
strong repressor of the epidermal cell
death response (Steffens & Sauter, 2005;
2009). In conclusion, symptoms of
bakanae disease including plant height
increase, inhibiting chlorophyll
formation, disruption of root growth and
susceptibility to infection are associated
with phytohormonal imbalance of GAs,
IAA and ABA (Quiz et al., 2015).
All
pathological, morphological, anatomical
traits and hormonal activities changes
due to bakanae infection and GA
3
treatment were highly matched and
reflected the same behavior. GZ 10101-
5-1-1-1 as a new promising line was
highly tolerant variety that can be used a
good donor for bakanae resistance in
breeding program.
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