Integrated Management of Ascochyta Blight on Chickpea Germplasm in Pakistan

Full text

Çukurova Tarım Gıda Bil. Der. Çukurova J. Agric. Food Sci.
37(1): 53-62, 2022
doi: 10.36846/CJAFS.2022.73
Research Article
Integrated Management of Ascochyta Blight on Chickpea Germplasm in
Pakistan
Hira NAWAZ
1
, Sadaf NAZ
2
, Amjad ABBAS
3
, Hefza AFZAL
4
, Waqas LIAQAT
5
,
Mukaddes KAYIM1*
ABSTRACT
Chickpea, an important pulses product of Pakistan, ranks 3rd among legumes in the world. The most important
fungal disease agent of chickpea Ascochyta rabiei is anthracnose, which causes 50 - 70% yield losses in
chickpeas. In this study, 10 chickpea genotypes inoculated with A. rabiei were screened. Under artificial
inoculum pressure, agronomic and physiological data were recorded. To manage this disease, the commercial
preparation of the biocontrol antagonist Trichoderma harzianum and some fungicides were then applied to
these genotypes. The resistivity of pathogen spores to T. harzianum antagonist and fungicides was tested in
vitro. While the chemical fungicides performed equal inhibition with the 1st and 2nd levels, the 3rd and 4th
levels of inhibition differed from each other. The biological antagonist commercial T. harzianum was found
to be effective in anthracnose disease. control.
Keywords: Antagonist, Ascochyta rabiei, Chemical management, Chickpea Biomass, Chickpea screening,
cell membrane stability.
Pakistan'da Nohut Germplazmında Ascochyta Blight'ın Entegre Mücadelesi
ÖZ
Pakistan'nın önemli bir bakliyat ürünü olan nohut, dünyada baklagiller arasında 3. Sırada yer almaktadır.
Nohut’un en önemli fungal hastalık etmeni Ascochyta rabiei nohutta %50 - 70 arasında verim kayıplarına
neden olan antraknozdur. Bu çalışmada A. rabiei ile inoküle edilmiş 10 nohut genotipinde tarama yapılmıştır.
Yapay inokulum ile agronomik ve fizyolojik veriler elde edilmiştir. Bu hastalıkla mücadele için, daha sonra
bu genotiplere biyokontrol antagonist Trichoderma harzianum ticari preparatı ve bazı fungisitler
uygulanmıştır. İn vitro koşullarda patojen sporlarının T. harzianum antagonisti ve fungistlere karşı dirençleri
test edilmiştir. Kimyasal fungisitler 1. ve 2. seviye ile eşit inhibisyon gerçekleştirirken 3. ve 4. seviye
inhibisyonda biribirinden farklılık göstermiştir. Biyolojik antagonist ticari T. harzianum preparatı hastalık
kontrolünde etkili bulunmuştur.
Anahtar Kelimeler: Antagonist, Ascochyta rabiei, Hücre zarı dayanıklılığı, Kimyasal mücadele,
Nohut biyokütlesi
ORCID ID (Yazar sırasına göre)
0000-0002-5396-6701, 0000-0001-7999-2222, 0000-0002-2417-3729, 0000-0001-6055-103X, 0000-0001-
6719-2340, 0000-0003-0309-0390
Yayın Kuruluna Geliş Tarihi: 21.02.2022
Kabul Tarihi: 05.05.2022
1
Department of Plant Protection, Faculty of Agriculture, Cukurova University, Adana, Turkey
2
Department of Microbiology, Faculty of Veterinary Sciences, University of Agriculture, Faisalabad, Pakistan
3
Department of Plant Pathology, Faculty of Agriculture, University of Agriculture, Faisalabad, Pakistan.
4
Agriculture Officer (PW& QCP) Jhang, Pakistan.
5
Department of Field Crop, Faculty of Agriculture, Cukurova University, Adana, Turkey.
*E-posta: kayimukaddes@gmail.com

Integrated Management of Ascochyta Blight on Chickpea Germplasm in Pakistan
54
Introduction
Chickpea (Cicer arietinum L.) is a self-
pollinated field crop that belongs to the
family fabeace. It is a diploid crop with 12
chromosomes. It is an important legume crop,
rich in protein, and an important part of a diet for
the vegetarian population of the world. It can
grow on a large agro-ecological range of climate
that’s why grown in more than 50 countries of
the world. (FAOSTAT, 2017; Tadesse et al.,
2017). It can grow in fertile to non-fertile soil
ranges. Chickpea is attacked by Ascochyta blight
disease which is caused by Ascochyta rabiei and
is considered as a major limiting factor of yield
worldwide (Jamil et al. 2010). The disease can
cause 40-70% losses if optimum conditions last
for 48 hours. (Malik and Bashir, 1984). Due to
epiphytotic occurrence, it causes complete crop
loss. (Pande et al., 2005). Symptoms appear on
the stem, leave, and pods of the plant which are
concentric lesions orange to brown. Blight can
be managed by several means like the use of
resistant varieties, cultural practices include
removal of diseased plants from the field
avoiding cultivation in the already infected field,
and crop rotation. (Lubian et al., 2019). In
Pakistan major part of south Punjab, consist of a
desert range where agriculture is based on
chickpea cultivation so crop rotation is not
applicable in this region. (GOP, 2017). Different
biocontrol agents and plant extracts are used to
manage the disease. (Hernandez-Terrones et al.,
2007; Khajista et al., 2011). Chemical fungicides
are also used to control blight disease in
chickpeas. (Pande et al., 2005). Biocontrol
agents like T. viride, C. globosum, and A.
implicatum were reported effective
against Ascochyta rabiei under In-
vitro conditions. (Bisen et al., 2020). Shafique et
al. (2011) evaluated the fungi toxic potential
of Tagetes erectus L. against A. rabiei the cause
of gram blight disease. At various concentrations
pathogen exposed (1, 2, 3, and 4% w/v) of
aqueous and methanol extracts of shoot and
flower of T. erectus using food poisoning
technique. Concentrations of both shoot and
flower extracts significantly suppressed the
growth target pathogen. Reduction of colony
diameter was 4-35% and 55-73% of A.
rabiei due to different concentrations of flower
and shoot extracts of T. erectus and 12-50% and
4-42% due to different compositions of the
methanolic shoot and flower extracts of T.
erectus respectively.
In this research, A. rabiei was isolated screened
on different cultivars along with a management
strategy using different chemical and biocontrol
agents.
Methodology
Isolation and purification of the fungal
pathogen
The pathogen usually infects seedlings and is
soil and trash borne in nature. Infected plant
tissues like stem, leaves, and pods were cut into
2cm pieces and then sterilized in a 2% aqueous
solution of hydrogen peroxide. The samples
were placed on media which were incubated for
20 days at 25°C in the incubator. (Walter, 2009).
Purification of the pathogen was done by
transferring mycelium using the hyphal tip
method and was identified morphologically by
using available literature especially based on
plate colour, colony pattern, presence of conidia,
spore shape, size, and structure by slides
preparation for microscopy (Keogh et
al., 1980; Barnett and Hunter, 1972).
1. Collection and sowing of germplasm
Chickpea was sown in sick plots (fungal
inoculum were given in soil and covered the
soil with polythene sheet for 48 hours)
present in the experimental area of the
Department of the Plant Pathology
University of Agriculture Faisalabad. Seeds
of 10 genotypes were sown in plots in four
replications. Germination percentage was
recorded in plots. A. rabiei was grown on
chickpea media. After 21 days, spores were
harvested by adding chilled water to the Petri
plate followed by sieving through four layers
of sterile muslin cloth. The spore of A.
rabiei (Figure 1) was counted under the light
microscope by using a hemocytometer. The
inoculum concentration was adjusted to 104
spores/ml. Artificial inoculum of 104
spores/ml was given near the roots and
sprayed on plants in such a way that it is
disturbed equally and similar treatment for

Integrated Management of Ascochyta Blight on Chickpea Germplasm in Pakistan
55
all plants. Different parameters in the
controlled and inoculated plants were
recorded like seed germination, germination
percentage of seed, number to flowering, no
of pods per variety flowering colour, and
seed weight.
Figure 1: Shows (a) A. rabiei (b) fruiting body of A. rabiei (c) Trichoderma harzianum (d)
Beauvaria bassiana isolate based on visual testing and microscopy.
2. Application of biocontrol agents
After one week of inoculation of the pathogen in
plots biocontrol agents which are B.
bassiana and T. harzianum was applied. They
were grown on PDA media at 25+ 2ºC for 2
weeks, 10 ml of chilled double distilled water
was added to the Petri plate containing
cultures and scratch with needle gently and then
filter with 4 folding of muslin cloth. Then these
spore suspensions were applied to plants in such
a way that T. harzianum + B. bassiana were
applied to the first replication. T. harzianum was
applied to the 2nd replication and B.
bassiana was applied to the 3rd replication and
4th replication was kept as control.
3. Physiological parameters
After inoculation of biocontrol agents, different
physiological parameters and biomass (Excised
leaf water loss, Relative water contents, Relative
dry weight of leaf, Cell membrane stability,
Root/shoot ratio, Grain yield) of an individual
plant. Such as Ali et al. (2011).
Excised leaf water loss
Chickpea plants were harvested, plants were
washed under running tap water to remove soil
from roots. Labelled plants according to their
variety and treatment and weight each plant.
This was the fresh weight of chickpea plants.
Place plants into a hot dry oven for 6 hours at
28ºC. After that weigh plants one by one and
again place them in a hot dry oven for 24 hours
at 70ºC. Weighted the dried plants, after
collecting all readings by using the formula
given below find out excised leaf water loss. It
was done according to the method followed by
Ali et al. (2009b).

Integrated Management of Ascochyta Blight on Chickpea Germplasm in Pakistan
56
ELWL = [(Fresh weight -Weight after 6 h)/
(Fresh weight - Dry weight)] ×100
Relative water content
Chickpea plants were harvested, plants were
washed under running tap water to remove soil
from roots. Labelled plants according to their
variety and treatment and weight each plant.
This was the fresh weight of chickpea plants.
Place plants into a tub containing water for 24
hours in the dark so that plants get strongly
turgid. Weighted plant and again place in a hot
dry oven for 48 hours at 78ºC. Again weighted
the dried plants, after collecting all readings by
using the formula given below to find out the
relative water content.
RWC (%) = (Fresh weight – Dry weight)/
(Turgor weight – Dry weight) × 100
Cell membrane stability
Take fresh leaflets from the chickpea plants of
each treatment. Wash each leaflet with deionized
double distilled water (D3water) and place in a
test tube containing 20 ml of D3 water and take
a reading of sample by EC meter (electron
conductivity) and place in an autoclave at 121ºC
and 15psi pressure after this take the reading
again with EC meter than using formula and
calculation find out Cell membrane stability.
CMS (%) = [(1-(L1/L2)] × 100
The relative dry weight of leaf
This was calculated by taking the weight of
ELWL dry weight and turgid weight and dry
weight from relative water content. The relative
dry weight of the leaf finds out by putting value
in the formula given below. Ali et al. (2009b).
RDW = Dry weight / (Turgor weight - Dry
weight)
Disease assessment and disease rating scale
As the crop was grown under natural inoculum
pressure, the crop plants were observed for
assessment of the disease. At various growth
stages like flowering and pod formation stages,
disease development was monitored and
recorded. A disease rating scale was adapted as
was already reported in the literature. This
disease usually appears at flowering and pods
stages. It was monitored at both stages to collect
valid information as reported (Farooq
et al., 2005). Disease grading was done in the
field and micro plots.
The disease was recorded visually and rated by
using the following scale given by
 Highly Resistant = Less than 1% of
plant wilted.
 Resistant = 1-10% of plants wilted.
 Moderately Resistant = 11-20% of
plants wilted.
 Susceptible = 21-50% of plants wilted.
 Highly Susceptible = 51% or more of
plants wilted. (Iqbal et al., 2005)
Disease severity:
Disease severity was calculated by the
following formula given by (Mehrotra and
Aggarwal, 2003).
Disease Severity = 𝐴𝑟𝑒𝑎 𝑜𝑓 𝑝𝑙𝑎𝑛𝑡 𝑡𝑖𝑠𝑠𝑢𝑒 𝑖𝑛𝑓𝑒𝑐𝑡𝑒𝑑
𝑇𝑜𝑡𝑎𝑙 𝑎𝑟𝑒𝑎 (𝑓𝑎𝑟𝑚)
Disease Incidence % = 𝐷𝑖𝑠𝑒𝑎𝑠𝑒𝑑 𝑝𝑙𝑎𝑛𝑡𝑠
𝑡𝑜𝑡𝑎𝑙 𝑛𝑜 𝑜𝑓 𝑝𝑙𝑎𝑛𝑡𝑠 ∗100
4. In-vitro management of A. rabiei pathogen
of chickpea:
In-vitro management of A. rabiei was done by
using various chemicals. For this purpose,
commercially available chemicals were used in
different concentrations. In this experiment, 12
chemicals were tested against single isolates
of A. rabiei. Seven concentrations were made
with 3 replications each in 96 well plates. Spores
of A. rabiei were harvested from a pure culture
grown on chickpea media in the Petri plate.
For in-vitro testing 96 well plates were used.
Liquid media @ 100µl was mixed with 50µl of
spore suspension @ 104 spores in this volume. A
liquid suspension of fungicides @ 50µl was
added to each well according to the
concentration of the plate. Seven different levels
(using serial dilution) of fungicides were used in
this experiment. Instead of chemicals, water was
used in the positive control. In the second control
spores and distilled water of the same volume
were used as control. A total volume of 200 µl in
each well was maintained. The chemical-treated
spores containing plates were kept at the same
temperature for incubation. After 24 hours the
growth of spores and their germination was
measured based on optical density at the

Integrated Management of Ascochyta Blight on Chickpea Germplasm in Pakistan
57
wavelength of 630 nm by using a
spectrophotometer at the department of Center
of Agriculture Biotechnology and Biochemistry
(Abbas et al., 2013).
Results and discussion
Chickpea plants were artificially infected with A.
rabiei and then after the appearance of
symptoms plants were treated with biocontrol
agents. All the physiological and biomass data
were collected and analyzed by using statistical
tools with a 0.05 level of significance. The
ANOVA table below represents the variance of
root length, shoot length, number of pods per
plant, and weight of grain from one plant. Along
with treatment, ten different varieties were used
therefore, the experiment contain two factors
which were treatments with varieties. Table 1
represents that root length, shoot length number
of pods per plant, and weight of grain from one
plant gives a higher level of significance with
treatment and varieties individually but in case
of interaction all were non-significant except
root length which favours the experiment as A.
rabiei doesn’t affect root system of the plant
.
Table 1: ANOVA table of physiological trait of treated chickpea plants. The significance level for
the trait was taken 5 % for comparison of mean value among selected genotypes
MSs: Mean sum of the square, **: Highly significant, *: Significant, N/A: Non-significant
Different physiological parameters were
observed with disease attack, varieties, and
biological treatments along with control. Results
are presented in the form of graphs, cell
membrane stability graphs show that treatments
showing similar lettering were statistically non-
significant. T. harzianum enhances the cell
strength of all the cultivars under observation in
contrast with control while least was observed in
the combination of biocontrol agents. Likewise,
the same pattern was somehow followed within
other parameters. In relative water, the content
was maximum with biocontrol combination as
well as B. bassiana in contrast with T.
harzianum and control. Excised leaf water loss
was maximum observed in the control treatment
and least were observed with T.
harzianum. Maximum chlorophyll content was
observed with T. harzianum in all varieties and
the least were observed with control treatment.
All the treatments showing different lettering
were statistically significant.
In-vitro management was done with 12
fungicides by measuring the OD of the 96 well
plates. Results are shown in the form of graphs
as well an ANOVA table is given below.
Graphical representation in figure 5 showed the
inhibition percentage with different levels of
each fungicide along with control after 96 hours
of the experiment. As fungicides were in higher
concentration in the first 2 levels that’s the
reason for maximum inhibition by all fungicides
with those levels. So it was observed that mic 50
is common for all fungicides at 3rd and 4th
levels. ANOVA Table 2 and 3 showed inhibition
of A. rabiei with level and time with 0.05% level
of significance. Results with levels and time
individually showed a higher level of
significance while in interaction with each other
it showed non-significant results for all the used
fungicides.
Source
Mean SS of
root length
Mean SS of
shoot length
Mean SS of No.
of pods per plant
Mean SS of the
weight of grain per
plant
Treatment
65.8692**
551.104 **
150.000**
260.962**
Variety
22.2993**
764.00 **
260.759**
87.2867**
Treatment*variety
7.9729**
7.681 N/A
4.946 N/A
3.683 N/A

Integrated Management of Ascochyta Blight on Chickpea Germplasm in Pakistan
58
Figure: 2 shows cell membrane stability (A), relative water content (B), excise leaf water content
(C), and chlorophyll content after inoculation of the pathogen (D) and B. bassiana and T.
harzianum individually and in combinations along with control.
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50
100
150
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Cell membrane stability
B. bassiana + T. harzianum B. bassiana T. harzianum control
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Pb-2008 K-01016 O9027 Noor-2009 10004 10036 k-01019 k-01014 9013 k-01007
Relative water content
B. bassiana + T. harzianum B. bassiana T. harzianum control
i
B
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Excised leaf water loss
B. bassiana + T. harzianum B. bassiana T. harzianum control
C
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Chlorophyll content
B. bassiana + T. harzianum T. harzianum B. bassiana control
D

Integrated Management of Ascochyta Blight on Chickpea Germplasm in Pakistan
59
Figure 3. Presenting inhibition percentage with 7 levels of different fungicides.
Table 2: ANOVA table of chemical inhibition on A. rabiei. ANOVA table for the significance level
for inhibition was taken 5 % for comparison of mean value among themselves
MSs: Mean sum of the square, **: Highly significant, *: Significant, N/A: Non-significant
Table 3: ANOVA table of chemical inhibition on A. rabiei. ANOVA table for the significance level
for inhibition was taken 5 % for comparison of mean value among themselves
Source
MSs
Metalaxial
mancozeb
MSs
Clipper
MSs
Tubeconazole
MSs
Cymoxial
mencozeb
MSs
Difenaconazole
MSs
Domalite
Level
0.24766**
0.20629**
0.03941**
0.69129**
0.18111**
0.42452**
Time
0.40352**
0.60638**
0.00011**
0.47521**
0.10575**
0.43815**
Level*Time
0.01951 N/A
0.02976N/A
0.00824 N/A
0.09217N/A
0.08823 N/A
0.00640 N/A
MSs: Mean sum of the square, **: Highly significant, *: Significant, N/A: Non-significant
df
a
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ab
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F
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t
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C
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JEG
AFF
K
HB
K
0
20
40
60
80
100
120 Inhibition percentage after 96hrs
Level 1 Level 2 Level 3 Level 4 Level 5 Level 6 Level 7
Source
MSs
Tropsin M
MSs
Thiomil
MSs
Sucess
MSs
Segawin
MSs
Sulphur
MSs
Curzate
Level
0.41706 **
0.07838**
0.05913**
0.19429**
0.44689**
0.39258**
Time
0.03614**
0.00211**
0.08367**
0.05240**
1.19480**
0.19039**
Level*Time
0.08148 N/A
0.00539 N/A
0.01302 N/A
0.24915 N/A
0.30035 N/A
0.01424 N/A

Integrated Management of Ascochyta Blight on Chickpea Germplasm in Pakistan
60
Figure 4 below represent the spore and mycelia
of A. rabiei after treatment with the chemical.
Slides were prepared from the broth media
present in 96 well plates to visualize the chemical
mode of the spores and mycelia of the fungus and
it has been clear that all the spores and mycelium
are de-shaped in the slides and mycelium become
the mass of debris in the end along with control.
Slides were stained with lactophenol and
observed under 40X to capture images.
Figure 4: Mycelium growth of A. rabiei with A and B are chemical treatment along with C as control.
Discussion
Ascochyta blight caused by Ascochyta rabiei in
chickpea. Its distribution varied depending upon
environmental factors and the amount of
inoculum in the field. A. rabiei is controlled by
using several chemical fungicides and biocontrol
agents worldwide. But the use of the chemical is
not economical as well as not eco-friendly
approach, it also makes pathogen resistant
(Pande et al., 2005).
Research conducted for testing several
chemicals against A. rabiei, it was noted that
chlorothalonil, zineb, captan, antracol,
propiconazole, penconazole, and thiabendazole
is effective and controlling the spread of
Ascochyta blight (Ahmad et al., 2021).
Likewise, biocontrol agents, T. viride,
Chaetomium globosum, and Acremonium
implicatum under In-vitro conditions proven
effective against A. rabiei (Bisen et al.
2020). Chickpea blight is controlled by Aliette
fungicide under in vitro conditions and causes
significant inhibition which supports the present
research. Chongo et al. (2003a) reported that the
application of chemicals at the right time is very
important to reduce the losses caused by A.
rabiei. The use of chlorothalonil at two different
stages reduce the incidence up to 8% which was
45% for the control treatment. Gan et al. (2006)
concluded that foliar application along with
integrated management is very effective for
disease management in chickpea, which
supports our research that plants with the proper
application of fungicide or correct time for
biocontrol helped in disease reduction. The use
of protective fungicides helps to keep away
disease pathogens from coming in
contact. Choice of good and effective fungicide
is very important. A mixture of foliar and
protective fungicides was used for the
experiment to find the effective one. Demirci et
al. (2003) tested chlorothalonil, azoxystrobin
under in-vitro and In-vivo conditions and found
that these two fungicides do not perform well on
the plate but under field conditions, they perform
very well against A. rabiei. Shtienberg et al.
(2000) concluded that protective fungicides like
zineb, Bordeaux mixture, captan are very
important in disease reduction but not effective
enough on susceptible cultivars. In recent years
number of new fungicides had been reported as
effective against A. rabiei. Effective fungicides
against A. rabiei are boscalid, pyraclostrobin,
difenoconazole, azoxystrobin, tebuconazole,
mancozeb which support our research because
several fungicides are part of current research
(Gan et al., 2006).
MacLeod and Galloway (2002) Mancozeb is
used in Australia, Canada, and Israel for the
control of chickpea blight. In the present
research, mancozeb performs well for blight
fungus. MacLeod et al. (2002) also found that
carbendazim which is now banned,
difenoconazole, and tebuconazole was tested in
India, the Western part of Asia, Australia, and
A
B
C

Integrated Management of Ascochyta Blight on Chickpea Germplasm in Pakistan
61
North of Africa, and these fungicides have
proven effective.
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