Journal of Phytopathology and Pest Management 9(1): 14-31, 2022
pISSN: 2356-8577 eISSN: 2356-6507
Journal homepage: http://ppmj.net/
∗
Corresponding author:
Mahmoud M. H. Hassanin,
E-mail: dr.hassanin.1978@gmail.com
14
Copyright © 2022
Chia charcoal rot disease and its management
using certain bio-agents in Egypt
Madian M. Mergawy1, Mahmoud M. H. Hassanin2*, Sarah A. A. Ahmed1, Abdallah A. M. Ali3
1Central laboratory of Organic Agriculture, Agricultural Research Center (ARC), Giza, Egypt
2Ornamental, Medicinal and Aromatic Plant Disease Department, Plant Pathology Research Institute,
Agricultural Research Center (ARC), Giza, Egypt
3Plant Pathology Department, Faculty of Agriculture and Natural Resources, Aswan University, Aswan, Egypt
Abstract
Keywords: chia, charcoal rot, Macrophomina phaseolina, Trichoderma asperellum, Streptomyces rochei.
The current study was performed to control the charcoal stem rot disease
caused by Macrophomina phaseolina in chia plants (Salvia hispanica L.).
Macrophomina phaseolina was morphologically identified as the causal
pathogen of charcoal stem rot on naturally infected chia plants showing typical
symptoms of disease and obtained from Fayoum, Giza, and Menoufia
governorates, Egypt in the 2021 cultivation season. Additionally, the molecular
characterization of the causal pathogen revealed 99.65-100% identity with
several isolates of the same species. The isolates obtained from all governorates
under study resulted in pre- and post-emergence damping-off with significant
variations. However, M. phaseolina isolated from Fayoum governorate
recorded the highest percentage of charcoal stem rot. Moreover, the filtrate of
the same isolate caused the highest percentages of wilted seedlings. The
presence of yeast, amonium chloride and urea as nitrogen sources resulted in
the loss of M. phaseolina mycelial color. The pigmented isolate of M. phaseolina
(Fayoum isolate) exhibited a high virulence to chia plants in greenhouse
compared to the non-pigmented one. The two bio-agents; Trichoderma
asperellum and Streptomyces rochei, isolated from the rhizosphere soil of
healthy chia plants, significantly inhibited M. phaseolina fungal growth in
comparison with the control in vitro. In greenhouse experiment, the fungicide
Tricyclazole was the most efficient application for reducing the incidence of
disease as well as increasing the plant growth measurements, i.e., number of
spikes per plant, number of branches and plant height, followed by the biocide
New-Actino. The combined use of the bio-agents T. asperellum and S. rochei
was greatly efficient in both decreasing the disease incidence and improving the
plant growth parameters compared to individual use of each. The current study
indicated the potential use of the biocide New-Actino, T. asperellum and S.
rochei as fungicidal alternatives for controlling chia charcoal rot disease.
Mergawy et al., 2022
15
1. Introduction
Chia (Salvia hispanica), belonging to the
family Lamiaceae, is cultivated for its edible
seeds. Chia seeds, which are rich in omega-3
fatty acids and fibre, receive attention for their
potential health advantages (Ayerza & Coates,
2011) and they are being cultivated for
commercial use in many countries, including
the US, Bolivia, Argentina, Australia, and
Peru. In Egypt, chia farms had indications of
charcoal stem rot, which reduced plant stand
and vegetative development. The causal
pathogen was found to be Macrophomina
phaseolina, a soil-borne fungus that has several
commercial hosts (Su et al., 2001). It is a
dangerous disease that reduces chia yield
(Nada, 2016; El-Kaed et al., 2021). Charcoal
stem rot disease could result in a reduction of
both seed quality and yield (Smith & Wyllie,
1999). Five hundred plant species belonging to
about 100 families are affected by the global
spread of M. phaseolina. Certain diseases
incited by the pathogen include seedling blight
and charcoal rot diseases (Ghosh et al., 2018;
Dhingra & Sinclair, 1978). The fungal
pathogen could significantly reduce the yields
of crops like sorghum and soybean when
temperatures are between 30–35 °C and soil
moisture is lower than 60% (Kaur et al., 2012).
In the worst-case situation, diseases that
developed in the pre-emergence stage of
groundnut cultivars have been known to cause
100% yield losses (Marquez et al., 2021).
Trichoderma spp. is known to improve
systemic acquired resistance and development
in plants. They also effectively control several
soil-borne fungal pathogens, such as M.
phaseolina (El-Kaed et al., 2021).
Trichoderma may exhibit biocontrol by using
many antagonistic actions, i.e.,
mycoparasitism, release of antibiotics, and
nutrition competitiveness. The genus
Trichoderma has several different species and
is considered to be a plant growth-stimulating
and promising biological control agent in
several crops (Savazzini et al., 2009; Bai et al.,
2008; Verma et al., 2007). The present study
was performed to investigate the charcoal stem
rot disease in chia plants incited by M.
phaseolina under Egyptian conditions and the
role of its black pigment in the virulence of this
disease, as well as the evaluation of different
fungicidal alternative means for controlling
this disease under greenhouse conditions.
2. Materials and methods
2.1 Isolation and identification of the
charcoal stem rot causal pathogen
Chia plants exhibiting the classic symptoms of
naturally occurring infection with charcoal
stem rot disease were obtained from Fayoum,
Giza, and Menoufia governorates, Egypt in the
2021 season. Following a tap water wash and
cutting into tiny pieces, the infected stems
were treated with 2% sodium hypochlorite for
2 min for surface sterilization. They were then
washed many times in sterilized distilled water
and placed on potato dextrose agar (PDA)
plates, followed by incubation for 7 days at
27±1°C. Based on the cultural and
morphological characteristics of the causal
pathogen, it was purified and identified
according to Lakhran et al. (2018). For
additional uses, purified cultures were placed
on slants of PDA and stored at 5 °C.
2.2 Isolation and purification of the fungal
biological agents
Rhizosphere soil samples of healthy chia
plants cultivated in fields extensively infested
with the causal agents of root-rot were
obtained from different governorates, i.e.,
Fayoum, Giza and Menoufia. As described by
Abd-El-Moity (1976), one g of rhizosphere
soil sample and 99 ml sterilized water were
mixed in a glass bottle, followed by using an
electric shaker for 1 h. The suspension was
then diluted to 10-4 and 1 ml was added to Rose
Mergawy et al., 2022
16
Bengal medium (Johnson et al., 1960) to
isolate the antagonistic fungi.
2.3 Isolation and purification of the
actinomycetes biological agents
One gram of rhizosphere soil sample and 19 ml
sterilized water were mixed in a flask to
prepare a 1/20 dilution, followed by a shake in
a rotary shaker (150 rpm). To avoid bacterial
and fungal contamination, two drops of the
aforementioned suspensions of each sample
were then individually transferred to a 25 ml
cylinder that contained 10 ml of a 1:140
dilution of phenol with water. Then, using the
method outlined by Crook et al. (1950) and
Waksman (1959), Petri dishes containing 15
ml of starch nitrate agar (SNA) medium (pH
7.2) were streaked with 0.1 ml of each soil
suspension. For every sample, five plates were
utilized, followed by incubation at 28°C and
routinely checked. Colonies of actinomycetes
were selected after 3-5 days according to their
morphological parameters, and they were
purified and grown on SNA medium.
2.4 Identification of the causal pathogen and
bio-agent isolates based on molecular
methods
The molecular characterization of M.
phaseolina, T. asperellum and S. rochei
isolates was performed at the Molecular
Biology Research Unit, Assiut University.
Potato sucrose agar (PSA) medium was used
for the cultivation of T. asperellum and M.
phaseolina, followed by incubation at 28°C for
5 days, as described by Pitt and Hocking
(2009). For S. rochei cultivation, 10 ml of
nutrient broth medium in sterile test tubes were
used, followed by incubation at 28°C for 48 h,
as described by Zimbro et al. (2009). DNA
extraction was carried out using Patho Gene-
Spin DNA/RNA extraction kit (Intron
Biotechnology Company, Korea). The primers
ITS1 F (5'-TCCGTAGGTGAACCTGCGG-
3'), and ITS4 R (5'-TCCTCCGCTTATTGAT
ATGC -3') were used in polymerase chain
reaction (PCR) to amplify ITS regions of the
rRNA gene of T. asperellum and M.
phaseolina. While the primers 27F (5’-
AGAGTTTGATCC TGGCTCAG-3’) and
1492R (5’-GGTTACCTTGTTACGACTT-3’)
were used to amplify the rRNA gene of S.
rochei, as described by (White et al., 1990).
The amplified products were purified before
being sequenced with the same primers used in
PCR amplification. The Basic Local
Alignment Search Tool (BLAST) available on
(NCBI) website was then used to analyze the
obtained sequences. Phylogenetic tree
construction was performed with MegAlign
(DNA Star) software version 5.05.
2.5 Pathogenicity tests
Three M. phaseolina isolates from the
governorates of Fayoum, Giza, and Menoufia
were used in the pathogenicity test. Separate
cultures of each isolate were made using
autoclaved sorghum grain medium (100 ml
water + 100 g sorghum + 50 g washed sand),
which were kept at 27±1°C for 15 days. Before
being used, sand-clay soil (1:1 w/w) was
treated with a 5% formalin solution and
allowed to dry for 14 days prior to usage. The
tested isolates were mixed at a rate of 1% with
the sterilized soil and put into 50-cm-diameter
pots. Additionally, pots were left without
infestation and used as uninoculated control.
To promote the colonization of the tested
fungal isolates, pots were watered until
completely submerged one week prior to
planting with 20 seeds/ pot. Four replicates
were used for each tested isolate and control.
Pre-emergence damping-off, post-emergence
damping-off and charcoal stem rot incidence
at 15, 30 and 60 days following planting,
Mergawy et al., 2022
17
respectively, were recorded as percentages
using the following formula noted by Waller et
al. (2002):
𝐷𝑖𝑠𝑒𝑎𝑠𝑒 𝑖𝑛𝑐𝑖𝑑𝑒𝑛𝑐𝑒 (%)=𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑖𝑛𝑓𝑒𝑐𝑡𝑒𝑑 𝑝𝑙𝑎𝑛𝑡𝑠
𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑝𝑙𝑎𝑛𝑡𝑠 × 100
After re-isolating of the fungal isolates from
the infected plants, they were compared to the
initial isolates.
2.6 Effect of M. phaseolina cultural filtrates
on chia seedlings
Three isolates of M. phaseolina from the
governorates of Fayoum, Giza, and Menoufia
were grown on Czapeks liquid medium. One
hundred ml of media in a 250-ml conical flask
was inoculated with a 5-mm-diameter disc of
M. phaseolina isolate (7-day-old), and it was
then incubated at 27 °C for two weeks. Flasks
without fungal inoculation were used as a
control. Cultures were sterilized with a
Millipore syringe filter (0.45 μm) after being
filtered by Whatman's No. 1 filter paper. Using
deionized water, five concentrations of fungal
filtrate for each isolate were made at rates of
20, 40, 60, 80, and 100%. Equal amounts of the
sterilized fungal filtrates with their
concentrations and the control medium
(uninoculated) were then transferred to glass
vials and planted with healthy chia seedlings
(30-day-old), 5 seedlings for each, and kept
under 12 h light and 12 h dark at 28-30 °C. For
each treatment and control, three vials were
used, and the percentage of wilted seedlings
was recorded at 72 h after inoculation, as
reported by Hassanin (2007).
2.7 Antagonistic effect of S. rochei and T.
asperellum on M. phaseolina mycelial
growth in vitro
In the current experiment, three isolates of T.
asperellum (from Fayoum, Giza, and
Menoufia governorates) and two isolates of S.
rochei (from Fayoum and Giza governorates)
as mentioned by Siddiqui et al. (2001) and
Abd-El-Moity (1985), were evaluated for their
antifungal effect on M. phaseolina isolate
(from Fayoum governorate) mycelial growth
in vitro. Starch Nitrate Agar (SNA) medium
[soluble starch (20 g), K2HPO4 (1 g), KNO3
(2g), MgSO4.7H2O (0.5 g), NaCl (0.5 g),
CaCo3 (3 g), agar (20 g), FeSO4.7H2O (0.001
g) and distilled water (1000 ml), (Waksman,
1959)] was used to study the antagonistic
impact of S. rochei on M. phaseolina. On the
other hand, Gliotoxin Fermentation Agar
(GFA) medium [Dextrose (25 g), KH2PO4 (2
g), ammonium tartrate (2 g), FeSO4.7H2O (0.1
g), agar (20 g), MgSO4.7H2O (1 g) and
distilled water (1000 ml), (Brian & Hemming,
1945)] was used to study the antagonistic
impact of T. asperellum on M. phaseolina.
Using the tested medium in each case, Petri
plates (9-cm-diameter) were inoculated at one
side with a disc of M. phaseolina, measuring
0.5 cm in diameter, which was obtained from
the outer edges of a culture that was 7 days old
on GFA medium. The opposite side of every
plate, in consideration of the tested medium,
was streaked with a loop from a 7-day-old S.
rochei culture grown on starch nitrate broth or
inoculated with a disc of T. asperellum (0.5-
cm-diameter), which was obtained from a 4-
day-old culture. As an untreated control, Petri
dishes were inoculated with M. phaseolina
only, which were replicated, as well as the
treated ones, three times. Following incubation
at 25°C and 27°C, in the case of the
antagonistic study with S. rochei and T.
asperellum, respectively, the percentage of the
linear growth reduction of M. phaseolina in
treated plates was calculated once the mycelial
growth had completely covered the control
plates medium surface, as follows:
Linear growth reduction (%) = 100 - [G2 / G1 × 100]
Mergawy et al., 2022
18
Where, G1= the mycelial growth diameter
(mm) of M. phaseolina in control plates.
G2= the mycelial growth diameter (mm) of M.
phaseolina in treated plates.
2.8 Effect of nitrogen sources on dark pigment
production in M. phaseolina mycelium
In this experiment, five different sources of
nitrogen were separately added to Czapek’s
medium, which contained [sucrose (30 g),
NaNO3 (2 g), MgSO4.7H2O (0.01 g), KCl (0.5
g), agar (15 g), K2HPO4 (1 g) and distilled
water (1000 ml)]. Nitrogen sources such as
yeast, ammonium chloride, urea, peptone, and
sodium nitrate were added to the medium
individually, instead of NaNO3, at a rate of 2
g/liter before sterilization. Medium of each
nitrogen source, as well as medium without
any nitrogen sources as a control, were poured
into Petri dishes and 3 replicates were used for
each. Using M. phaseolina discs selected from
the outer edges of 7-day-old culture that was
isolated from Fayoum governorate and grown
on PDA media, dishes were inoculated and
then incubated for 7 days at 27 °C. The
morphological characters of the tested isolate
were visionary examined, as mentioned by Ali
et al. (2017).
2.9 Greenhouse experiments
2.9.1 Effect of the fungal dark pigment
production on M. phaseolina aggressiveness
on chia plants in greenhouse
In this investigation, both pigmented and non-
pigmented colonies of M. phaseolina (Fayoum
isolate) were cultivated on autoclaved
Czapek’s liquid medium supplemented by two
distinct sources of nitrogen: sodium nitrate and
yeast, for 15 days at 27°C. Before being used,
sand-clay-peatmoss soil (1:1:1 w/w/w) was air
dried for 14 days after being sterilized for one
week with a 5% formalin solution. Following
that, the soil was mixed separately with the
pigmented isolates of M. phaseolina and the
non-pigmented one at a 1% ratio (w/w) and it
was put into 50-cm-diameter pots. For
comparison, pots were left without fungal
inoculation and used as a control. To promote
fungal colonization, pots were watered until
completely submerged one week prior to
planting with 20 chia seeds/ pot, with four
replicates for each treatment and control. The
incidence of disease was recorded at 15, 30
and 60 days following planting as a percentage
of pre-emergence damping-off, post-
emergence damping-off and charcoal stem rot,
respectively, using the following formula:
𝐷𝑖𝑠𝑒𝑎𝑠𝑒 𝑖𝑛𝑐𝑖𝑑𝑒𝑛𝑐𝑒 (%)=𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑖𝑛𝑓𝑒𝑐𝑡𝑒𝑑 𝑝𝑙𝑎𝑛𝑡𝑠
𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑝𝑙𝑎𝑛𝑡𝑠 × 100
2.9.2 Controlling chia charcoal stems rot using
different treatments under greenhouse conditions
Two biological agents individually or in
combination, T. asperellum and S. rochei were
prepared as a suspension at a rate of 20 ml/l
water after being adjusted to a concentration of
30 × 106 cfu/ml, biocide: New-Actino
(Streptomyces griseorubes and Trichoderma
hamatum; produced at Central Laboratory of
Organic Agriculture, Agricultural Research
Center (ARC), Giza, Egypt) at a rate of 20 ml/l
water and Fungicide: Tricyclazole WP
[Common name: Tricyclazole. Chemical
composition: 5-Methyl-1,2,4-triazolo{3,4-b}-
(1,3) benzothiazole. Manufacture: Elanco
Products Co., Div. of Eli Lilly and co.] at a rate
of 1 g/L water (according to Hassanin, 2013)
were evaluated for controlling chia charcoal
stem rot disease incited by M. phaseolina
(Fayoum governorate) in greenhouse.
Trichoderma asperellum isolate (Fayoum
governorate) was cultured in liquid Gliotoxin
Fermentation Medium at 25°C for 11 days in
total darkness, as described by Abd-El-Moity
and Shatla (1981), while liquid starch medium
Mergawy et al., 2022
19
was used for growing S. rochei isolate
(Fayoum governorate) at 30°C ±2 for 7 days.
Treatments were applied two times as soil
drench: at the sowing date and 15 days
following the sowing. Pots without treatments
were used as an untreated control and 20 chia
seeds/pot (50-cm-diameter) were planted with
four replicates for each treatment and control.
Using the previously mentioned formula, pre-
emergence damping-off, post-emergence
damping-off and charcoal stem rot incidence
were calculated as percentages at 15, 30, and
60 days after sowing, respectively. The plant
growth measurements, i.e., number of
branches, plant height, as well as spike
number/ plant were also recorded.
2.10 Statistical analysis
Experiments were carried out using a totally
randomized design. Using SAS software,
version 2004, data were subjected to statistical
analysis of variance in accordance with methods
described by Snedecor and Cochran (1980).
3. Results
3.1 Disease symptoms
As shown in Figure (1), charcoal stem rot
disease symptoms on naturally infected chia
plants begin on stems as little black spots that
enlarge to cover the entire stem. Subsequently,
the disease may cause significant losses in chia
yield.
3.2 Molecular identification of the causal
pathogen and bio-agent isolates
The amplified PCR products of DNA obtained
from M. phaseolina, T. asperellum and S.
rochei were submitted to GenBank after being
sequenced using the selected primers with
accession numbers (OP861486 for M.
phaseolina, OP164570 for T. asperellum and
OP164572 for S. rochei).
Figure 1: Naturally infected chia plants with charcoal stem rot disease compared to healthy
plants. A (Healthy control plants) and B (infected plants).
Macrophomina phaseolina strain AUMC15577
showed 99.65-100% identity with several
strains of the same species (Figure 2). The
phylogenetic tree also showed Phoma
herbarum as an outgroup strain. Trichoderma
asperellum strain AUMC15576 showed
Mergawy et al., 2022
20
99.67-100% identity with several strains of the
same species (Figure 3). Streptomyces rochei
strain AUMC-B471 showed 99.93-100%
identity with several strains of the same species
(Figure 4). Bacillus subtilis is also included in
the phylogenetic tree as an outgroup strain.
3.3 Pathogenicity tests
Table (1) presents the pathogenicity test of
three isolates of M. phaseolina, which were
isolated from naturally infected chia plants
obtained from Fayoum, Giza and Menoufia
governorates. All the tested isolates of M.
phaseolina resulted in pre-emergence
damping-off and post-emergence damping-off
with significant variations. Nevertheless, Fayoum
isolate showed the greatest percentage of charcoal
stem rot (10%) at 60 days after planting, which
subsequently decreased those of healthy
survival plants to the lowest percentages
(32.92%). In contrast, the lowest percentages
of pre-emergence damping-off, post-
emergence damping-off and charcoal stem rot
(28.33 and 12.08% and 5.41%, respectively)
resulted from M. phaseolina isolated from
Menoufia governorate. Subsequently, it
reduced the healthy survival plants to 54.18%.
Figure 2: A phylogenetic tree based on ITS sequences of the rDNA of M. phaseolina strain
AUMC15577 (Accession: OP861486) matched closely similar strains obtained from the GenBank. B
(Botryosphaeria), M (Macrophomina) and P (Phoma).
Mergawy et al., 2022
21
Figure 3: A phylogenetic tree based on ITS sequences of the rDNA of T. asperellum strain
AUMC15576 (Accession: OP164570) matched closely similar strains obtained from the GenBank.
Figure 4: A phylogenetic tree based on 16S sequences of S. rochei strain AUMC-B471 (Accession:
OP164572) matched closely similar strains obtained from the GenBank.
3.4 Effect of fungal filtrates on percentages
of wilted chia seedlings
As shown in Figures (5 and 6), all the tested
fungal filtrates increased the percentage of
wilted seedlings after 72 h from treatment
when compared to their controls. The tested
concentrations of fungal filtrates were shown
to be positively correlated with the
percentages of wilted seedlings. In this regard,
M. phaseolina filtrate of Fayoum isolate at
concentrations of 80 and 100% recorded the
highest percentages of wilted seedlings
(100%) (Figure 5). Additionally, its lower
Mergawy et al., 2022
22
concentration (20%) increased the percentage of wilted seedlings (26.7%) compared to the control.
Table 1: Pre-, post-emergence damping-off and chia charcoal stem rot percentages incited by M.
phaseolina at 15, 30 and 60 days after sowing, respectively.
Governorate
Disease incidence (%)
At seedling stage (15 and 30 days after sowing)
At maturity stage (60 days after sowing)
Post-emergence
Charcoal stem rot (%)
Survived plants (%)
Fayoum
27.08
10.00
32.92
Giza
15.41
8.30
42.96
Menoufia
12.08
5.41
54.18
Uninoculated control
0.0
0.0
100.0
L.S.D. at 5%
0.631
0.665
0.985
3.5 Antagonistic effect of S. rochei and T.
asperellum on M. phaseolina mycelial
growth in vitro
In this investigation, three isolates of T.
asperellum (from Fayoum, Giza, and
Menoufia governorates) and two isolates of S.
rochei (from Fayoum and Giza governorates)
were evaluated for their antagonistic effect on
the M. phaseolina (Fayoum governorate)
mycelial growth. All the tested isolates of
Trichoderma and Streptomyces significantly
inhibited the fungal growth of M. phaseolina
compared to the untreated control.
Figure 5: Effect of fungal filtrates of M. phaseolina isolates on percentages of wilted chia seedlings
after 72 hours of inoculation.
0
13.3
26.7
46.7
66.7
86.7
0 0
13.33
26.7
46.7
73.3
0
26.7
66.7
86.7
100 100
0
20
40
60
80
100
120
020 40 60 80 100
Percentages of wilted chia seedlings (%)
Fungal filtrate concentrations (%)
M. phaseolina (Giza) M. phaseolina (Menoufia) M. phaseolina (Fayoum)
Mergawy et al., 2022
23
Figure 6: Effect of M. phaseolina cultural filtrates at various concentrations on chia seedlings
compared to control.
The highest antagonistic effect was obtained
by T. asperellum treatment (T1, isolated from
Fayoum governorate), which recorded 60.1%
reduction in linear growth of M. phaseolina
(Table 2, Figure 7). Following that, S. rochei
(ST1, isolated from Fayoum governorate)
inhibited M. phaseolina linear growth to
54.9% (Table 2, Figure 8). On the contrary, S.
rochei (ST2, isolated from Giza governorate)
showed the least effect against M.
phaseolina, with 35.8% inhibition in linear
growth.
Figure 7: Antagonistic effect of T. asperellum on the growth of M. phaseolina in vitro. T1
(Fayoum isolate), T2 (Giza isolate) and T3 (Menoufia isolate).
Figure 8: Antagonistic effect of S. rochei on growth of M. phaseolina in vitro. ST1 (Fayoum
isolate) and ST2 (Giza isolate).
Mergawy et al., 2022
24
Table 2: Antagonistic effect of T. asperellum and S. rochei isolates on M.
phaseolina mycelial growth.
Bio-agents
Reduction* in M. phaseolina mycelial growth (%)
Trichoderma asperellum (T1, Fayoum isolate)
60.1
T. asperellum (T2, Giza isolate)
50.3
T. asperellum (T3, Menoufia isolate)
48.1
Streptomyces rochei (ST 1, Fayoum isolate)
54.9
S. rochei (ST 2, Giza isolate)
35.8
Control
0.0
L.S.D. at 5%
0.958
*Reduction compared to the control treatment.
3.6 Effect of nitrogen sources on dark
pigment production in M. phaseolina
mycelium (Fayoum isolate)
Figure (9) shows the great impact of altering
any of the nitrogen sources on the morphological
characteristics of M. phaseolina mycelial growth
including color and density. As shown, the presence
of yeast, amonium chloride and urea resulted in the
loss of M. phaseolina mycelial color.
Figure 9: Effect of nitrogen sources on mycelium color of M. phaseolina (Fayoum isolate): A
(Control), B (Pepton), C (Sodium nitrate), D (Urea), E (Amonium chloride) and F (Yeast).
3.7 Effect of the M. phaseolina pigment on
the aggressiveness of M. phaseolina on chia
plants in greenhouse
Table (3) shows that the pigmented isolate of
M. phaseolina (Fayoum isolate) was higher
than the non-pigmented one in their virulence
to chia plants, as they showed the greatest
percentages of pre-emergence damping-off
and post-emergence damping-off, 36.67 and
13.33% at 15 and 30 days after sowing,
respectively. Additionally, it showed the
greatest percentage of charcoal stems rot
(8.33%) at 60 days after planting, which
subsequently decreased those of healthy
survival plants to the lowest percentages
(41.67%).
3.8 Using different treatments for
controlling chia charcoal stem rot under
greenhouse conditions
3.8.1 Effect on disease incidence
As shown in Table (4), all treatments were
efficient in decreasing the disease incidence.
However, the fungicide Tricyclazole was the
best treatment for decreasing the disease
incidence to the lowest percentage (1.25%),
subsequently; it recorded the highest
Mergawy et al., 2022
25
percentage of survived plants (93.75%).
Following that, the biocide New-Actino
reduced the disease incidence to 5% and
subsequently resulted in increases in the
percentages of survived plants (88.75%). The
obtained results demonstrate that using
biological agent isolates in combination was
more efficient in managing the disease than
using individual ones. In this regard, S. rochei
treatment recorded the highest percentage of
pre-emergence damping-off, post-emergence
damping-off and disease incidence (16.25,
17.50 and 11.25% respectively) compared to
the other treatments, while the percentages
decreased when it was applied in combination
with T. asperellum (7.5, 6.25 and 8.75%,
respectively). Generally, using any of the
biological agents led to a significant reduction
in pre-, post-emergence and charcoal stem rot
incidence and subsequently resulted in
increases in the percentages of survived plants
compared to the control.
Table 3: Pre-, post-emergence damping-off and chia charcoal stem rot percentages caused by
the pigmented and non-pigmented M. phaseolina isolates at 15, 30 and 60 days after planting,
respectively.
Fungus
Pigmentation of the
fungal cell wall
Damping-off (%)
Charcoal stem rot
(%)
Survived plants
(%)
Pre-emergence
Post-emergence
Macrophomina phaseolina
(Fayoum)
Pigmented
36.67
13.33
8.33
41.67
Non pigmented
8.33
3.33
3.33
85.01
Uninoculated control
0.00
0.00
0.00
100.00
L.S.D. at 5%
1.639
0.831
0.576
1.345
Table 4: Impact of different treatments on the incidence of chia charcoal rot under greenhouse
conditions.
Treatments
Disease incidence (%)
At seedling stage (15 and 30 days after sowing)
At maturity stage (60 days after sowing)
Pre-emergence
Post-emergence
Charcoal stem rot (%)
Survived plants (%)
Trichoderma asperellum
10.00
13.75
10.00
66.25
Streptomyces rochei
16.25
17.50
11.25
55.00
T. asperellum + S. rochei
7.5
6.25
8.75
77.50
New-Actino
2.5
3.75
5.00
88.75
Tricyclazole
2.5
2.5
1.25
93.75
Untreated control
37.50
25.00
27.50
10.00
L.S.D. at 5%
0.828
0.982
0.946
0.995
3.8.2 Effect on plant growth parameters
Data in Table (5) shows that all tested
treatments enhanced plant height, number of
branches and number of spikes per plant in
comparison with the untreated control. Nevertheless,
Tricyclazole was the best treatment, which
increased plant height (cm), no. of branches
and number of spikes/plant to the highest percentages
(52.25, 6.50 and 7.25, respectively). Following that,
the biocide New-Actino increased the plant
growth parameters to 46.00, 5.00 and 6.5,
respectively. The combined use of the biological
agents was better than the individual use for
improving the plant growth parameters. In this
regard, the treatment of T. asperellum increased the
plant growth parameters to 36.75, 3.50 and 4.25,
respectively, while the combined treatment of
T. asperellum and S. rochei increased them to
40.75, 4.25 and 5.00, respectively.
Mergawy et al., 2022
26
Table 5: Impact of various treatments on chia vegetative growth.
Treatments
Plant height (cm)
Number of branches
Number of spikes/plant
Trichoderma asperellum
36.75
3.50
4.25
Streptomyces rochei
33.25
3.00
3.25
T. asperellum + S. rochei
40.75
4.25
5.00
New-Actino
46.00
5.00
6.50
Tricyclazole
52.25
6.50
7.25
Untreated control
25.75
1.25
1.75
L.S.D. at 5%
1.408
0.391
0.303
4. Discussion
In the current study, samples of naturally
infected chia plants had classical symptoms of
charcoal stem rot disease were collected from
Fayoum, Giza, and Menoufia governorates.
The disease symptoms begin on stems as little
black spots that enlarge to cover the entire
stem. The symptoms described by Misaka
(2019) seem a lot like those of the infected
plants. As mentioned by Kaur et al. (2012), the
pathogen can spread from diseased roots to
stems, blocking the vascular tissues in the tap
root. It is additionally capable of spreading to
seeds, decreasing germination and resulting in
seedling rot. The causal pathogen's molecular
characterization in the present research
matched 99.65-100% with many strains of the
same species. It has been shown that specific
primers aimed at the ITS region may
specifically identify a number of significant
agricultural fungi, as reported by Edel et al.
(2000) and Druzhinina et al. (2005). In
comparison to the uninoculated control, the
isolates from all governorates under study
produced significant variations in pre- and
post-emergence damping-off. Nonetheless, the
highest percentage of charcoal stem rot was
caused by M. phaseolina isolated from the
Fayoum governorate. Furthermore, the highest
percentage of wilted seedlings was induced by
the filtrate of the same isolate. Melanin
pigment may be the cause of M. phaseolina's
aggressiveness, as reported by Polak (1990),
who mentioned that some soil fungi's melanin
pigment is a key factor in determining their
pathogenicity. Our obtained results are also in
accordance with those mentioned by Nada
(2016), who reported that M. phaseolina was
among the most destructive fungi on chia
plants. Our findings are consistent with El-
Garhy's (1994) findings, which indicated that
M. phaseolina culture filtrates produced
browning and damaging of the veins in lentil
leaf tissues in addition to necrotic areas. Our
results reveal the significant effects of
changing any of the nitrogen sources on the
morphological characteristics of M.
phaseolina mycelial growth, such as color and
density. These findings indicate that variations
in the media's nitrogen source and structure
may have an impact on the fungal pathway.
These results are in accordance with those
mentioned by Pihet et al. (2009), who reported
that sequencing of the genes related to the
melanin production pathway revealed a
genetic abnormality in the early stages of this
pathway for Aspergillus fumigatus isolates.
Also, our results are in accordance with those
obtained by Ali et al. (2017). In vitro studies,
the mycelial growth of M. phaseolina was
significantly inhibited by the two bio-agents,
T. asperellum and S. rochei, which were
extracted from the rhizosphere soil samples of
healthy chia plants. There is a clear variation
in the antagonistic effect of the two tested bio-
agents as expressed in the obtained results,
which reveal that T. asperellum treatment (T1,
isolated from Fayoum governorate) recorded
the highest percentage reduction in M.
phaseolina mycelial growth. The quantity and
number of antifungal compounds that the bio-
Mergawy et al., 2022
27
agent produces might be the cause of these
differences, as reported by Harman et al.
(2004). According to Abd-El-Moity (1976)
and Harman et al. (2004), Trichoderma spp.
interacts with the pathogen by a variety of
mechanisms, including mycoparasitism and
the synthesis of trichodermin and gliotoxin. In
several studies, Actinomycetes that produce
chitinase enzyme have been used as biological
control agents (Loqman et al., 2009; Prapagdee
et al., 2008; Sharifi et al., 2007). In greenhouse
experiment, the pigmented isolate of M.
phaseolina was shown to be more virulent
against chia plants than the non-pigmented
isolate, as proven by its greatest pre- and post-
emergence damping-off percentages as well as
the incidence of charcoal stem rot. These
results are supported by those obtained by
Pihet et al. (2009), who noted that the fungal
isolates with black pigments may be more
virulent because they protect the fungus from
host immune responses. Although other colors
may occasionally occur, the majority of dark
pigments in nature that are thought to be
melanin are typically either black or brown in
color, as reported by Cerenius and Söderhäll
(2004). Our results are also in agreement with
those reported by Ali et al. (2017). The most
effective treatment for both decreasing the
incidence of disease and improving the plant
growth measurements in greenhouse was the
fungicide Tricyclazole. The role of
Tricyclazole 75%WP in disease resistance was
studied by Ali et al. (2017), who mentioned
that Tricyclazole 75%WP at 500 and 1000 ppm
entirely suppressed the hyphal color of M.
phaseolina in vitro study. The authors also
proved the role of fungal black pigment in
increasing M. phaseolina aggressiveness to
cassia plants under greenhouse conditions. In
another investigation by Wheeler et al. (2004),
the authors mentioned that Tricyclazole 75%
WP in PDA cultures blocked melanin
synthesis by the Monosporascus cannonballus
wild types. The biocide New-Actino treatment
followed Tricyclazole for decreasing the
incidence of disease and improving the plant
growth measurements. As reported by
Hajieghrari et al. (2008) and Poovendran et al.
(2011), Trichoderma strains have a variety of
biocontrol mechanisms, including
mycoparasitism, competition, antibiosis,
hyphal contacts, and enzyme release, to
prevent infections caused by plant diseases.
Additional reports also explain this impact by
stating that T. harzianum secretes a variety of
enzymes (chitinase, protease, β-1,3-glucanase,
and cellulase) and antibiotics into the medium,
where they break down the pathogen's cell
wall and are essential for microparasitism
(Ait-Lahsen et al., 2001; Lorito et al., 1993;
Elad et al., 1982; Papavizas & Lumsden,
1980). Streptomyces rochei, belonging to the
actinomycetales order, is a valuable source of
bioactive secondary metabolites with
industrial and commercial applications, as
mentioned by El-Tarabily et al. (2000) and
Bressan and Figueiredo (2005). According to
Doumbou et al. (2001), actinomycetes are a
source of bioactive compounds, organisms
that promote plant development, and tools for
bio-controlling plant diseases. Many
antibiotics known to be effective against
phytopathogenic fungi have been isolated
from Streptomyces (Remsing et al., 2003;
Rodríguez et al., 2002; Hwang et al., 2001;
Kim et al., 1999). When compared to their
separate applications, the combined use of the
bio-agents T. asperellum and S. rochei
significantly improved the plant growth
parameters and reduced the disease in
greenhouse experiment. These findings are in
accordance with those mentioned by
Ezziyyani et al. (2007), who reported that
pepper root rot incited by Phytophthora
capsici can be reduced more effectively by
combining T. harzianum and S. rochei than by
applying them alone. Also, Schmidt et al.
Mergawy et al., 2022
28
(2004) reported that the erratic nature of
biological control must be lessened by the
application of many biocontrol agents.
5. Conclusion
Macrophomina phaseolina was characterized,
depending on morphological and molecular
methods, as the causal agent of charcoal rot
disease in naturally infected chia plants
showing typical symptoms of disease and
obtained from Fayoum, Giza, and Menoufia
governorates in the 2021 cultivation season.
Changing any of the nitrogen sources was
found to be highly effective on the
morphological characteristics of M.
phaseolina mycelial growth, such as color and
density, which subsequently influenced the
isolate's aggressiveness against plants in
greenhouse. The combined use of the bio-
agents T. asperellum and S. rochei, isolated
from the rhizosphere soil of healthy chia
plants, was highly effective for both reducing
the disease incidence and improving the plant
growth parameters, as well as the biocide New-
Actino, under greenhouse conditions.
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