Journal of Phytopathology and Pest Management 7(1): 91-108, 2020
pISSN: 2356-8577 eISSN: 2356-6507
Journal homepage: http://ppmj.net/
∗
Corresponding author:
Mohamed A. Eliwa,
E-mail: mohamedeliwa.5419@azhar.edu.eg
91
Copyright © 2020
Evaluation of different chemicals to control
Erysiphe
betae
the causal pathogen of sugar beet powdery mildew
Mohamed M. El-Sheikh Aly
1
, Ali H. ElShaer
2
, Anwar A. Galal
3
, Harbi M. Abd-Alla
3
, Mohamed A. Eliwa
1*
1
Agricultural Botany Department, Faculty of Agriculture, Al-Azhar University, 71524 Assiut, Egypt
2
Plant Pathology Research Institute, Agricultural Research Center, Giza, Egypt
3
Plant Pathology Department, Faculty of Agriculture, Minia University, Minia, Egypt
Abstract
Keywords: sugar beet, powdery mildew, Erysiphe betae, macronutrients, fungicides.
92
1. Introduction
Sugar beet (
Beta vulgaris
L.), is an
herbaceous dicotyledonous plant belongs
to family Amaranthaceae (formerly
Chenopodiaceae). It is considered as one
of the two major sugar crops around the
world and an important crop of temperate
climates which provides nearly 40% of
the world’s annual sugar production and
is a source for bioethanol and animal feed
(Bastas and Kaya, 2019). Under field
conditions, several pathogenic fungi
attack sugar beet plants causing serious
diseases
i.e
., Cercospora leaf spot
(
Cercospora beticola
Sacc.) and rust
(
Uromyces betae
Press). Sugar beet
powdery mildew which is caused by
Erysiphe betae
(Vanha) Weltzien is
among the most important foliar diseases
of sugar beet worldwide (Gobarah &
Mekki, 2005). The disease is
economically significant for growers
worldwide and can cause sugar yield
losses up to 30 % (Francis, 2002). Gado
(2013) reported that powdery mildew is
considered as a major foliar disease of
sugar beet in areas with dry and relatively
warm weather conditions throughout the
world, devastating foliar disease affecting
plant growth and consequently sugar
production. Egyptian environmental
conditions help the fungus to spread
rapidly specially in the late sowings after
September. Losses in sucrose could reach
82.9 % for some cultivars due to powdery
mildew infection. Total soluble solids
percent and root weight were
dramatically affected by disease severity
under infected conditions (El-Fahhar,
2008).
Grimmer et al
.
(2007) reported
that if the disease is not controlled it can
cause a 20 to 35 percent loss in sugar
yield. Crop loss is due to a reduced root
yield and often to a lower concentration
of sugar in roots. Both effects apparently
are due to a reduced efficiency of
diseased leaves and to their premature
death, when roots are rapidly enlarging.
As part of the environment, nutrients
influence plant, pathogen and microbial
growth to remain an important factor in
disease control. The interaction of
nutrition in these components is dynamic
and all essential nutrients are reported to
influence the incidence or severity of
some diseases, mineral nutrients are the
components of plants and regulate
metabolic activity associated with
resistance of a plant and virulence of a
pathogen. Adequate nutrition is generally
required to maintain a high level of
disease resistance. Nutrient sufficiency
also may shorten a susceptible growth
stage for some plant-pathogen
interactions
(Huber & Haneklaus, 2007).
Macronutrients are well recommended as
fungicide alternatives for enhancing plant
health, subsequently inducing plant
resistance and controlling the disease in
parallel with their safe influence on
human health (Huber & Haneklaus,
2007). Control of sugar beet powdery
mildew is mainly achieved by
applications of broad spectrum systemic
fungicides
(Byford, 1996).
Although, the
wide spread use of the chemical
fungicides has become a subject of
research concern due to their harmful
effect on non-target organisms as well as
their possible carcinogenicity (Ziedan &
Farrag, 2011). However, further studies
should concern safe, applicable, reliable
and efficient replacement of chemical
fungicides by other safer chemical or
natural compounds harmless to plants or
human health. The objectives of this
study were to (1) investigate the spread
of sugar beet powdery mildew disease in
some governorates in Upper Egypt and
(2) to assess the role of some different
chemical compounds on reducing
powdery mildew disease incidence on
sugar beet.
93
2. Materials and methods
2.1 Survey of sugar beet powdery
mildew
Survey of sugar beet powdery mildew
was conducted in different districts of
two Governorates (3 districts) namely
Abnob, Dayrot and Manfalot (Assiut
Governorate) and Maghagha, Samallot
and AbuQurkas (El-Minia Governorate),
Egypt. At least 3 fields of each district
were concerned. Each field under survey
was determined with a field map, 5
sampling sites were designated per field
tested and one of each of the four corners
plus one in the center of the field.
Sampling sites were located at least 5
meter from the edge of the field (Ray &
McLaughlin, 1942). Severity of powdery
mildew was monitored 4 times at 20
days’ intervals. Area under disease
progress curve was conducted.
2.2 Powdery mildew disease
assessment
Evaluation of disease severity was
accomplished by examining both sides of
leaves and rating disease intensity as the
extent of leaf area covered by the fungus
mycelium on a scale of 0 to 4. Disease
severity was determined according to the
scale by Whitney et al. (1983). Scale
ranged from 0- 4 categories whereas 0=
no mildew colonies observed, 1=1-25%,
2=26-50%, 3=51-75% and 4=76-100% of
matured leaf area covered by mildew.
Area Under Powdery Mildew Progress
Curve (AUPMPC) was calculated for the
assessment period using the following
equation adopted by Chiha et al. (1997):
AUPMPC = D (1/2 (Y
1
+Y
k
) + (Y
2
+Y
3
k-1
)
Where: D= Time interval; Y
1
= First
disease score; Y
k
= Last disease score;
Y
2
and Y
3
= Intermediate disease score.
2.3 Estimating conidia survival
Greenhouse experiment was conducted
in 3 m × 3 m area well-isolated protected
one to study how long the time of
powdery mildew conidia still able to
attack sugar beet plants and initiate the
disease. Only powdery mildewed sugar
beet leaves were collected from the most
susceptible cv. FD.0807 grown under
open field and dried carefully on
sterilized benches with 70 % ethyl
alcohol to avoid rottenness, then packed
in plastic bag and stored at room
temperature until used. Two sugar beet
cultivars Sirona and F.D.0807 were sown
(4 seeds /pot) in 30 cm diameter plastic
pots, filled with sterilized sandy clay soil,
nine pots for each cultivar in three
replicates, each replicate consisted of
three pots and nine pots of each cultivar
which were isolated apart by thin white
plastic sheets served as control. Artificial
inoculation was done 60 days from
planting by shaking dried diseased sugar
beet leaves (after five months of storage)
over the growing plants at a height of
about 30cm. Disease severity was
estimated after seven days of inoculation.
2.4
In vitro
conidia germination tests
Conidia germination test was carried out
using light microscope, slides were
washed in 50 percent alcohol and wiped
with a cloth to remove inert particles in
order to prevent condensation of free
moisture on the glass surface at very high
relative humidity levels. The conidia
were detached and collected by
94
vigorously shaking infected leaves over
the glass slides placed at the bottom of a
plastic container 20×20×10 cm
3
. In order
to reduce variation in germination and to
obtain reproducible results, only 24 h old
conidia were utilized. For this purpose,
the plants were shaken every day to
prevent accumulation of old and
shriveled spores. Each slide was placed
on a U-shaped glass rod in a moist
chamber made up of sterile Petri dish
lined with filter paper saturated with
sterile distilled water. Petri dishes were
incubated at 25±2°C (Awad et al., 1990)
for 24 h before examination. One set of
chambers was kept in the light and
another in darkness. Three slides were
used as replicates for each particular
treatment. The percentage of germination
was based on the following formula:
2.5 Scanning electron microscopic
examination
Sample preparation: In order to study the
three dimensional structure through
scanning electron microscope (SEM) of
the haustoria and the infection mode on
the susceptible cultivar FD.0807, fresh
infected leaves (120 days old, 48 h after
symptom appearance) were collected, put
in paper bag and carried to Electron
Microscope Unit at Assiut University,
Egypt. The leaves were cut into
appropriate samples and were subjected
to fixation in 5 % cold buffered
gluteraldehyde for 2 days. The samples
were then washed by cacodylate buffer
for three times thirteen minutes for each
and post fixed in 1% osmium tetroxide
for 2 h. Samples were then washed in
cacodylate buffer for three times thirteen
minutes each and then dehydrated by
using ascending series of ethanol 30, 50,
70, 90 for 2 h, 100 % for two days, and
then to amyl acetate for two days.
Critical point drying was applied to the
samples by using liquid carbon dioxide.
Each sample was sticked on metallic
blocks by using silver paint. By using
gold sputter coating apparatus, samples
were evenly gold coated in a thickness of
15 nm (Bozzola et al.
,
1991).
2.6 Control of powdery mildew disease
Experiments were carried out at the
experimental field of the Faculty of
Agriculture, Al-Azhar University, Assiut,
Egypt during 2014/2015 and 2015/2016
growing seasons. Resistant and
susceptible sugar beet cultivars Sirona
and FD.0807, respectively were selected
for the experiments of powdery mildew
disease control. Field plots consisted of
two rows (9 m long and interspace
between plant and another 20 cm) and
arranged in a split plot design with three
replicates per treatment. One plot was
specified for one tested compound and
one plot was left for control. Field was
fertilized and irrigated as usual. Plants
were thinned to one plant /hole and left
for natural infection. Large area around
the plots was left without treatment to
avoid any contamination by any treated
chemicals from neighboring fields
(Gado, 2013).
2.6.1 Time of application
Treatment applications were started 105
days after sowing (the first sign of the
disease has appeared). Plants were
sprayed five times during each season
with 20 days’ intervals. Disease severity
95
was determined (5 times) in order to
evaluate treatments after ten days from
each time of spraying of tested
compounds. Solutions of each tested
compounds were applied using a hand
sprayer, at a volume of 2 liters of tap
water per plot (until run off). Thirty
plants were used for each treatment.
Plants without spraying were served as
control. AUPMPC values were calculated
as described before.
2.6.2 Estimation of total soluble solids
(TSS) percentage and root weight of
the treated sugar beet plants
At harvest, three replicate samples, each
sample of thirty roots for five sprays
treatments were randomly collected for
determination of root weight and sugar
analysis. Juice analysis were done by
using a digital refractometer to determine
TSS % of root juice and a precise hand
scale was used to measure root weight.
2.6.3 Effect of applying macronutrients
on the disease severity
Three chemical compounds containing
macronutrients
i.e.
calcium chloride,
potassium silicate and sodium
bicarbonate were tested to study their
effect against powdery mildew disease of
sugar beet plants Sirona cv. and
F.D.0807 line. Each compound was used
as foliar spraying at the concentrations of
0.1, 0.2 and 0.3 g /l. Sugar beet plants
were sprayed after 105, 125, 145, 165
and 185 days from sowing date. Bellis®
38 % WG Fungicide was used as a
comparative treatment which applied in
the dosage (0.5 g/l) as cited in its user
manual sheet as recommended by the
manufacturer (BASF™).
2.6.4 Effect of fungicides
In the study, the used fungicides were
Bellis 38 %
(25.2 % w/w boscalid and
12.8 % w/w pyraclostrobin), Collis 30 %
(20 % w/v boscalid and 10 % w/v
kresoxim–methyl), Camzin 50 %
(50 %
w/w carbendazim), Tilt 25 %
(25 % w/v
Propiconazole) and Permatrol 99 %
(99
% v/v Jojoba oil). Fungicides were
applied at the recommended dosage as
summarized in Table (1).
Table 1: Trade name, group name, chemical group, common name, recommended doses and
production Company of tested fungicides.
Trade name
Group name
Chemical group
Common name
Recommended
dose
Production
company
Bellis® 38% WG
Succinate dehydrogenase
inhibitors
Pyridine-
carboxamides
Boscalid
50 g/l
BASF™
Quinone outside Inhibitors
Methoxy-
carbamates
Pyraclostrobin
Collis® 30% SC
Succinate dehydrogenase
inhibitors
Pyridine-
carboxamides
Boscalid
50 ml/l
BASF™
Quinone outside Inhibitors
Oximino-acetates
Kresoxim-methyl
Camzin® 50% WP
Methyl Benzimidazole
Carbamates
Benzimidazoles
Carbendazim
75 g/l
CAM™
Tilt® 25% EC
DeMethylation Inhibitors
Triazoles
Propiconazole
15 ml/l
Syngenta
™
Permatrol™ 99% Oil
---------
-------
Jojoba oil
1000 ml/l
Soiltech™
96
2.6.5 Disease reduction
Disease reduction percent was calculated
according to
Ismail et al. (2012) as
follows:
2.7 Statistical analysis
Analysis of variance of the data was
carried out on the mean values of the
tested treatments according to the
procedures described by Gomez and
Gomez (1984). The least significant
difference (LSD) at 5% probability was
used for testing the significance of the
differences among the mean values of the
tested treatments for each character.
3. Results
3.1 Survey of sugar beet powdery
mildew
Data in Table (2) represent the survey of
powdery mildew disease which took
place in Assiut and Minia governorates
during 2012/2013 growing season. Data
showed that the highest AUPMPC value
was detected in Abnob locality followed
by Manfalot then Dayrot while, the
lowest AUPMPC value was found in
Maghagha locality followed by
AbuQurkas and Samallot localities
respectively.
3.2 Survival of conidia
Greenhouse experiment was conducted
on 2014/2015 growing season to
determine the overwintering capability of
vegetative mycelia and conidia of
Erysiphe betae
and their role in the
dissemination of the fungus. Obtained
results from consecutive observations
confirmed that the conidia collected from
the previous season (2013/2014) could
not initiate any type of infection or
disease symptoms which means that the
conidia could not survive as long as it
were stored in this experiment and the
conidia that remains on the crop debris at
the end of the season are not one of the
means which used by the fungus for its
overwintering.
Table 2: Area under powdery mildew progress curve (AUPMPC) values on sugar beet
plants (Glorius, Sirona and Samba cultivars) grown in different districts of Assiut and El-
Minia governorates, Egypt.
Governorate
District
Cultivar
AUPMPC
Assiut
Abnob
Glorius
660 ±1
Dayrot
Sirona
322 ±1
Manfalot
Samba
442 ±1
Minia
Maghagha
Sirona
118 ±1
Samallot
Glorius
190 ±1
AbuQurkas
Samba
228 ±1
Mean
------------
327 ±1
LSD at 0.05
53.9
3.3
In vitro
conidia germination tests
The purpose of this experiment was to
make a preliminary study on the
germination percentages of
E. betae
conidia on glass slides at 100 % relative
97
humidity at room temperature (25±2°C),
in light and in darkness. As shown in
Table (3) the percent germination in
darkness was lower than in light. A high
percentage of germinating conidia
formed appressorium on dry glass slides.
One appressorium was formed by the
germ tube of each conidium. The
appressorium formation was not affected
by light or darkness. The conidia of
Erysiphe betae
germinated at a fast rate
within 8 to 10 h of incubation. In the
same time 100 % relative humidity (RH)
was sufficient enough to prevent conidia
from shriveling.
Table 3: In vitro conidia germination percent at
light and darkness conditions.
Incubation conditions
Germination (%)
Light
74
Darkness
58
LSD at 0.05
18.27
3.4 Scanning electron microscope
examination
First observation on the scanning electron
microscopy (SEM) images of the
infection method of sugar beet with
powdery mildew pathogenic fungus
E.
betae
is that it penetrated the epidermis
of the leaves by the haustoria as shown in
Figure (1). On upper leaf surface, a great
amount of conidia was visible and
haustoria as well. Outer surface of the
haustoria is rough and wavy. The
haustoria are folded in many patches
forming a complex web which almost
completely covers the leaf. Haustoria
penetrated the leaf as a drilling machine
resulting at the side parts pieces of
mesophyll which are folded at the bottom
of the haustoria. It was clearly observed
that haustoria penetrated the stomata of
the leaf easily and successfully. The
convoluted haustoria penetrated the leaf
epidermis in many points infecting the
entire leaf surface. The haustoria were
convoluted and folded in multiple ways.
They entered perpendicularly the leaf
from the top. There were visible several
conidiphores formed, preparing new
source of secondary infection. The
entering zone of the haustoria, the hyphal
part is thickened, being like a connection
tube between the fungus and the leaf.
3.5 Effect of applying macronutrients
on disease severity of sugar beet
powdery mildew
Three compounds containing
macronutrients were tested for their
ability to control powdery mildew
disease on sugar beet. Data in Table (4)
showed that all the tested macronutrients
significantly reduced AUPMPC values
when sugar beet plants were sprayed
with them. It was noticed that increasing
macronutrients concentration sub-
sequently increased resistance of sugar
beet plants against powdery mildew
disease. The lowest (AUPMPC) on both
cultivars Sirona and FD.0807
respectively was achieved by 0.3 g/l of
sodium bicarbonate followed by 0.3 g/l
of calcium chloride and 0.2 g/l of sodium
bicarbonate respectively. The highest
(AUPMPC) was obtained by 0.1 g/l of
potassium silicate. The best treatment
(0.3 g/l sodium bicarbonate) was higher
in (AUPMPC) value than the tested
fungicide Bellis® 38 % WG.
98
Figure 1: Scanning microscope of haustoria and conidia where a: mature conidiospore freshly separated,
initiating germination tube turned towards a stoma on the adaxial leaf surface, note the root-like
appendages at the bottom trying to attach itself to the leaf, b: detached mycelial fragment with single
haustorium in the middle and about to enter a stoma, c: a network of hyphae on the abaxial surface of leaf
48 h after inoculation, successful penetration into the mesophyll (1), single haustorium seeking for stoma
(2) and fresh separated conidiospore ready for germination (3), d: magnifying successful penetration into
the mesophyll, e: one single haustorium about to enter an opened stoma on the abaxial leaf surface (1) and
two short conidiophores, each has single aged shriveled conidiospore at the top (2) and f: Enlarged view of
haustorium actually penetrated the mesophyll; using physical pressure is clear from the bending backward
resulted in visible folding in the upper side of the haustorium.
Table 4: AUPMPC values of sugar beet cultivars Sirona and FD.0807 as affected by macro nutrients
foliar spray under field conditions during 2014/2015 and 2015/2016 growing seasons.
Treatment
Conc. (g/l)
AUPMPC
cv. Sirona
cv. FD.0807
2014/2015
2015/2016
Mean
Disease
reduction (%)
2014/2015
2015/2016
Mean
Disease
reduction (%)
Calcium chloride
0.1
574
552
563 ±1
19.3
842
851
846 ±1
48.8
0.2
472
464
468 ±1
32.9
687
702
694 ±1
58
0.3
360
354
357 ±1
48.8
618
606
612 ±1
62.9
Mean
468
456
462 ±1
33.71
715
719
717 ±1
56.58
Potassium silicate
0.1
651
630
641 ±1
8.1
1070
1068
1069 ±1
35.3
0.2
582
597
589 ±1
15.6
922
910
916 ±1
44.5
0.3
514
495
504 ±1
27.7
801
772
786 ±1
52.4
Mean
582
574
578 ±1
17.16
931
916
923 ±1
44.11
Sodium bicarbonate
0.1
541
530
536 ±1
23.2
855
819
837 ±1
49.3
0.2
430
410
420 ±1
39.8
665
686
675 ±1
59.1
0.3
339
298
318 ±1
54.4
608
582
595 ±1
64
Mean
436
412
424 ±1
39.15
709
695
702 ±1
57.5
Bellis® 38% WG
78
68
73 ±1
89.5
191
138
165 ±1
90
Control
721
675
698 ±1
0.0
1652
1653
1653 ±1
0.0
LSD at 0.05
Treatment (T)
29.3
20.2
-----
-----
24.3
16.9
-----
-----
Concentration (C)
7.1
24.5
-----
-----
16.4
15.5
-----
-----
(T × C)
15.8
54.7
-----
-----
36.8
34.7
-----
-----
3.6 Estimation of total soluble solids
(TSS) contents of sugar beet plants
treated with macronutrients
Data in Figure (2) showed that the TSS
percentage in the roots of infected sugar
beet plants (Sirona and FD.0807
cultivars) and treated with the
compounds containing microelements
was increased significantly by all used
compounds as compared to control.
Sodium bicarbonate achieved the highest