Journal of Phytopathology and Pest Management 8(1): 29-45, 2021
pISSN: 2356-8577 eISSN: 2356-6507
Journal homepage: http://ppmj.net/
∗
Corresponding author:
Ahmed B. Mohamed,
E-mail: ahmedmohamed.5419@azhar.edu.eg
29
Copyright © 2021
Antifungal activity of bioagents and plant extracts
against certain fungal diseases of potatoes
Ahmed B. Mohamed
*
, Mohamed M. El-Sheikh Aly, Rafeek M. I. El-Sharkawy
Agricultural Botany Department, Faculty of Agriculture, Al-Azhar University, 71524 Assiut, Egypt
Abstract
Keywords: potato, Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium spp., bioagents.
Twenty-six fungal isolates were obtained from potato plants and tubers growing in
different localities in Egypt. The isolates were identified as 11 Rhizoctonia solani, 8
Sclerotinia sclerotiorum and 7 Fusarium spp. The 26 isolates were screened due to their
pathogenic capabilities and the most pathogenic isolate among each of the three
obtained genera was selected for this study. In vitro studies included the effect of 7
bacterial isolates, 6 Trichoderma isolates, as well as 6 plant extracts at four rates of
application against the three fungal pathogens, Trichoderma harzianum (T5) achieved
the highest mycelial growth inhibition, followed by T. asperellum (T34) and T.
harzianum (T10) isolates. Additionally, Bacillus subtilis (BS2) recorded the best mycelial
growth inhibition against the three tested fungi, followed by B. subtilis (BS1) and
B.megatirum(BM2). On the subject of plant extracts, garlic extract gave the greatest
reduction of the mycelial growth with all rates of application, followed by henna and
ginger extracts. Field experiments were conducted during 2018/2019 and 2019/2020
growing seasons to evaluate bioagent activities as well as plant extracts in reducing
disease severity caused by the three fore-mentioned pathogenic fungi. Trichoderma
harzianum (T5) exhibited the highest disease reduction in vivo, followed by (T34) and
Pseudomonas fluorescens (PF2), as compared with the control. Under greenhouse
conditions, garlic extract decreased disease severity of both Fusarium sp and S.
sclerotiorum, followed by henna and ginger extracts. On the other hand, henna extract
came in the first order in reducing disease severity caused by R.solani, followed by
ginger and garlic, as compared with the control. On the whole, Trichoderma harzianum
(T5) and T. asperellum (T34) were the best treatments, those reduced diseases severity to
the greatest extent if compared with the other treatments and the control.
Mohamed et al., 2021
30
1. Introduction
Potato (
Solanum tuberosum
L.) is one of
the most important crops in Egypt as well
as all over the world and produces a tuber
very rich in starch that it ranks as the
world’s fourth most paramount food crop,
after maize, wheat and rice (Cunnington,
2008). Annual production of potato in
Egypt during 2019 was about 5078347
tones (FAOSTAT, 2019). Potato crop is
susceptible to diseases caused by several
fungi, bacteria and other pathogens,
leading to considerable losses in yield
and quality (Khan et al., 2008; Walter et
al., 2001). Potato plant was infected by
Rhizoctonia solani
, which causes black
scurf of potato tubers, belong to
commonly appearing potato pathogens.
Scleroses of the mentioned fungi
occurring on sets can be the source of
infection for plants and descendant
tubers. Moreover, it makes their quality
worsen (Ahrenniemi et al
.,
2005). Potato
yield losses caused by this disease
amounted even to 50 % (Häni et al
.,
1998). Besides, rhizoctoniosis constitutes
distinct aesthetic defect, which decreases
market value of potatoes intendent for
consumer purpose or for food industry
(Lutomirska, 2007).
Sclerotinia
sclerotiorum
is an ascomycetous
phytopathogenic fungus, which can infect
over 400 plant species in the world
(Boland and Hall, 1994). The pathogen
causes white mold in many potato
producing areas of Egypt, mainly in fields
irrigated by sprinkler systems (Ojaghian,
2009). Although a few reports are
available showing severe damage of
white mold on potato, the pathogen
frequently causes substantial yield losses
in fields (Ojaghian, 2011). The first
infection of potato plants by
S.
sclerotiorum
is initiated by ascospores
which, can infect only senescent tissues
(Atallah and Johnson, 2004). The
secondary spread of the pathogen
resulted from direct contact between
healthy and infected tissues, when the
pathogen colonized onto lower branches
and leaves (Abawi and Grogan, 1979).
Dry rot was caused by a number of
Fusarium
spp affecting sprouting and
emergence at the beginning of the season,
which results in yield losses and damage
to the quality of daughter tubers,
especially during storage (Borca and
Carmen, 2013; Al-Mughrabi, 2010;
Corsini and Pavek, 1986; Hooker, 1981).
Biological control of dry rot has been
demonstrated in laboratory studies using
bacterial antagonists (Kiewnick and
Jacobsen, 1997; Schisler et al., 1997;
Schisler and Slininger, 1994; Slininger et
al., 1994). The renewed interest in
biocontrol among agriculture biologists is
due to its eco-friendly protection against
weeds, insects, and plant diseases, a long-
lasting effect, and safety features. Some
of the bacterial antagonists, however,
also have been found to show direct
growth promoting effects on crop plant
inoculants (Deshwal
et al.
2003; Barker
and Paulitz, 1996). Studies on the
mechanisms of disease control by plant
extracts revealed that their biologically
active constituents may have either direct
antimicrobial activity (Amodioha, 2000;
Ansari, 1995) or induce host plants
defense response resulting in reduction of
disease development (Schneider and
Ullrich, 1994). Natural plant extracts
have been found effective against a wide
range of plant pathogens (Feng and
Zheng, 2007; Amodioha, 2003; Wilson
et
al.
1997).
This work aimed to study the
effect of biological control and different
plant extracts against several fungal
diseases of potato which lead to
considerable losses in yield and quality.
Mohamed et al., 2021
31
2. Materials and methods
2.1 Isolation and identification of the
causal pathogens
For isolation of fungal pathogens,
collected samples of potato plant and
tubers were washed carefully under
running tap water to remove the adjacent
soil particles, followed by sterile water.
Infected parts (stems or tubers) were cut
using sterilized scalpel into small pieces,
surface sterilized by dipping in 70% ethyl
alcohol for 2 minutes, dried well between
two sterilized filter paper, then
transferred to plates contained PDA
medium. Petri dishes were incubated at
25°C for 5 to 7 days, daily inspected for
fungal growth. The developed fungal
colonies were picked on PDA medium
and purified using hyphal tip or single
spore techniques. The purified fungi were
identified according to fungal
morphological and microscopical
characteristics as described by Barnett
and Hunter (1986), Booth (1977) and
Sneh et al
.
(1991).
2.2 Pathogenicity tests
The pathogenic capability of the isolated
fungi was conducted under greenhouse
conditions in the Farm of Faculty of
Agriculture, Al-Azhar University (Assiut
branch), Egypt during 2017/2018
growing season.
2.3 Inoculum preparation
The fungal inoculum was grown in 250
ml jars containing the following substrate
per jar (75 g grain barley, 25 g coarse
sand and 25 ml tap water to cover the
mixture in each jar). The jars were
autoclaved at 121°C for 30 minutes, left
to cool, then inoculated with the tested
fungi and incubated at 25°C for 15 days
to obtain sufficient growth of each
fungus. Then, sterilized plastic pots in
5% formalin solution (40 cm in diameter)
were filled with sterilized soil with
formalin solution at 5% (10 Kg /pot).
After that, the inoculum was mixed with
the soil at the rate of 3% (w/w) of soil,
then irrigated three times a week before
sowing to ensure even distribution and
growth of each particular fungus. Other
sterilized pots were filled with sterilized
soil and un-infested with the tested fungi
were kept as control. Three seeds pieces
were planted in each pot and three pots
were used as replicates for each
treatment.
2.4 Disease assessment
Black scurf disease severity was
determined by using 0- 5 grade scale
based on the percent of tuber surface
showing disease symptoms, where 0 = no
symptoms on potato tubers; 1 = less than
1 % tuber area affected; 2 = 1-10 % tuber
area affected; 3 =11-20 % tuber area
affected; 4 =21-51 % tuber area affected;
5 = 51 % or more tuber area affected,
according to Ahmad et al
.
(1995). Dry
rot disease severity was determined
visually using a 0-5 scale where "0"
represented no disease symptoms and a
"5" represented 100% dry rotted tissue.
(Schisler et al
.,
2000). White mold
disease severity was estimated as
following., 0-4 scale of where: 0 = 0
percent, 1 = 1–25 percent, 2 = 26–50
percent, 3 = 51–75 percent and 4 = 76–
100 percent of the surface area of the
shoot with symptoms of white mold,
according to Morton and Hall (1989).
Mohamed et al., 2021
32
2.5 Evaluation of antagonistic bacteria
against the pathogenic fungi
in vitro
The antagonistic bacteria were obtained
from MERCIN, Faculty
of Agriculture,
Ain Shams University, Egypt. The used
bacteria in this study were two isolates of
Bacillus subtilis
(BS1 and BS2), two
isolates of
B. megaterium
(BM1 and
BM2), one isolate of
Penibacillus
polymyxa
(BP
)
as well as two isolates of
Pseudomonas fluorescens
(PF1 and PF2).
These isolates were tested against the
pathogenic
fungi
R. solani, S.
sclerotiorum
and
Fusarium.
sp
in vitro.
The antagonistic
bacteria were grown on
nutrient sucrose agar medium (NSA),
which
consisted of peptone 5gm, beef
extract 3gm, sucrose 5gm, yeast extract
2gm, agar 20gm and distilled water per/
Liter for 5 days at 25±2°C. A 5mm
mycelial disc in diameter of the
pathogenic fungus taken, of advancing
zone of growing hyphae was transfered at
the center of 9 cm diameter Petri plates
containing PDA medium. On the other
hand, the antagonistic bacteria were
streaked at a distance of 2-3 cm either in
semicircular pattern. The culture plates
were incubated at 25±2°C and inhibition
zone was checked after 24, 48, 72 and 96
hours. Three replicate plates were used
for each treatment. The inoculated plates
with tested fungi only were served as
control. Percentage of growth inhibition
of the pathogen was calculated, when
pathogen achieved full growth in the
check by the following formula:
Mycelial growth inhibition (%) = (A−B /A) × 100
Where: A= The diameter of the mycelial
growth in control. B= The diameter of
the mycelial growth in treated Petri
plates.
2.6 Evaluation of antagonistic activity
of
Trichoderma
isolates
against the
pathogenic fungi
in vitro
The antagonistic.
Trichoderma
isolates
and the tested pathogenic fungi
R. solani
,
S. sclerotiorum
and
Fusarium
sp.
were
grown on PDA medium for 7 days at
25±2°C to study the efficacy of
Trichoderma
bioagents against the
pathogenic fungi. Discs (5 mm in
diameter) from each isolate of
Trichoderma
were cut and placed on
PDA medium in one side of Petri dish
and the opposite side was inoculated with
the pathogenic fungal isolates. Three
replicate plates were used for each
treatment. The inoculated plates with the
tested fungi only, without
Trichoderma
isolates served as control. The inoculated
Petri plates were incubated at 25±2°C
until fungal growth of control grew to
full Petri plates. At the same time, the
fungal growth was measured in two
dimensions. The percentage of mycelial
growth inhibition was calculated
according to the following formula:
Mycelial growth inhibition (%) = (A−B /A) ×
100
Where: A= The diameter of the mycelial
growth in control. B= The diameter of
the mycelial growth in treated Petri
plates.
2.7 Evaluation of antagonistic bacteria
against the tested fungi under
greenhouse conditions
Pot experiments were carried out during
2018/2019 and 2019/2020 growing
seasons to study the antagonistic effect of
the antagonistic bacteria against fungal
pathogens causing the diseases of potato
Mohamed et al., 2021
33
foliage and tubers (Cara cultivar) caused
by the pathogenic fungi under
greenhouse conditions. Bacterial isolates
were applied as soil treatment, 15 days
before planting by adding 100 ml of
bacterial suspensions (10
8
cfu /ml) for
each pot, which previously infested with
the pathogenic fungi. to study the effects
of the selected fowr antagonistic
bacteria.,
Penibacillus. polymyxa
(BP
), B.
subtilis
(BS2),
B. megaterium
(BM1) and
Pseudomonas
fluorescens
(PF2) for
controlling potato disease incidence. Cara
cv. were planted (3 seed tubers /pot) in
infested soil with the pathogenic fungi as
mentioned before. After 100 days from
planting, diseases severity was recorded.
The inoculum of
Trichoderma harzianum
isolates (T5, T7, T10 and
Trichoderma
asperellum
(T34) were used
as
antagonistic fungi against
R. solani, S.
sclerotiorum
and
Fusarium
sp
which
grown on barley medium as mentioned
before in the pathogenicity tests. The
antagonistic
Trichoderma
bioagents and
the fungal pathogens were mixed with
sterilized soil at the rate of 3% (w/w) of
soil for each antagonistic and pathogenic
fungi, 15 days before planting. Each pot
was planted with 3 potato seeds (Cara
cv.). The infested pots with the
pathogenic fungi only served as control.
Each treatment was replicated three
times. Percentage of potato disease was
calculated after 100 days as previously
mentioned in the pathogenicity tests.
2.8 Comparative effectiveness of plant
extracts on potato disease incidence
2.8.1 Preparation of plant extracts
Fifty grams from each leaf, or parts of
garlic cloves and rhizomes of Basil, Blue
gum, Garlic, Henna, Thyme and Ginger
were washed several times with tap
water, rewashed with sterilized distilled
water and left to air dry at room
temperature. Then, the plant parts were
cut into small pieces and crushed
separately in a porcelain mortar with 100
ml of ethyl alcohol (70%). The grinded
material was passed through six layers of
cheesecloth and Whatman filter paper
No. 1. The filtrate was centrifuged at
3000 rpm for 10 minutes and the
supernatant was filtered with sterilized
centered glass funnel (El- Shaer, 1998).
Four dilutions were prepared from each
crude extract using sterilized distilled
water. The dilutions were 5, 10, 15 and
20% culture filtrates.
2.8.2 Effect of different concentrations
of plant extracts on the mycelial
growth inhibition of the pathogenic
fungi
in vitro
Different concentrations of Basil, Blue
gum, Garlic, Henna, Thyme and Ginger
extracts prepared as previously
mentioned were used to study their
effects on the mycelial growth inhibition
of
R. solani, S. sclerotiorum
and
Fusarium
sp.
Dilution of an extract was
separately mixed with PDA medium
before solidification, then poured in
sterilized Petri dishes. Three replicate
plates were used for each concentration.
Petri plates were inoculated at the center
with equal disc, 5mm in diameter, taken
from 7 days old culture of any of the
tested pathogens. The Petri plates were
incubated at 25°C. Sterilized distilled
water was mixed with the same dilutions
on PDA medium instead of any extract
served as control (Singh et al., 1986).
The mycelial growth inhibition of the
Mohamed et al., 2021
34
tested fungi was measured after the
growth completely covered the control
plates by taken middle of two axis of
growth. The reduction of mycelial growth
inhibition was calculated using the
following formula:
Mycelial growth inhibition (%) = (A−B /A) × 100
Where: A= The diameter of the mycelial
growth in control. B= The diameter of
the mycelial growth in treated Petri
plates.
2.8.3 Effect of mixing plant extracts
with the infested soil on the disease
incidence under greenhouse conditions
Leaves, cloves and rhizome extracts of
basil, blue gum, garlic, henna, thyme and
ginger were used to study their efficacy
on controlling potato diseases under
greenhouse conditions. This experiment
was carried in the Farm of Fac of
Agriculture, Al-Azhar University (Assiut
branch), Egypt during 2018/2019 and
2019/ 2020 growing seasons. The plant
extracts were added to the infested soil
with the pathogenic fungi at the rate 3%
(w/w) as fore mentioned described. Two
different concentrations of plant extracts
i.e.
15 and 20% (v/w) of soil were added
and poured in the upper layer of the
infested in pots (40 cm in diameter).
Three potato seeds of Cara cv. were
planted in each pot. Three replicate pots
were used for each treatment. The
infested soil with the pathogenic fungi
and without any additive of plant extracts
served as control. The experiment was
irrigated and fertilized regularly. Data
were recorded after 100 days from
planting as disease severity as previously
mentioned.
2.9 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 (L.S.D) 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
and Discussion
3.1 Isolation and identification of the
associated fungi with the infected
potatoes
Different fungal isolates representing
three genera
i.e. R. solani, S.
sclerotiorum
and
Fusarium
sp
.
were
isolated from potato plants and tubers
collected from different
locations of
Menofeya, Behera and Minia
governorates, Egypt. The fungal isolates
were identified by
using the
morphological features of mycelium and
spores as described by
Barnet and Hunter
(1986), Booth (1977) and Sneh et al
.
(1991) and confirmed by Agric. Botany
Department, Faculty of Agriculture, Al-
Azhar University (Assiut Branch), Egypt.
3.2 Pathogenicity tests
Twenty-six fungal isolates were tested to
study their pathogenic capabilities on
potato plants and tubers of (Cara cv.)
under greenhouse conditions during 2018
growing season. Data in Table (1)
illustrated that all tested fungal isolates
were able to infect potato seedlings
Mohamed et al., 2021
35
causing black scarf, white mold and dry
rot diseases. All tested isolates
significantly increased the infection
potato compared with the control.
R.
solani
(R8), (R1),
S. sclerotiorum
(S4)
and
R. solani
(R4), gave the highest
disease severity as reached 71.73, 68.93,
64 and 58.36 %, respectively. On the
other hand, isolates
S. sclerotiorum
(S3),
Fusarium
sp. (F2),
S. sclerotiorum
(S5),
R. solani
(R6), followed by
S.
sclerotiorum
(S1), Showed moderate
disease severity as reached 51.8, 48.13,
45.8, 44.7 and 44 %, respectively While
R. solani
(No. 5, 7 and 10) and
Fusarium
sp. (No. 5 and 4), gave the lowest disease
severity as reached 15.2, 20.5, 21.5, 22.4
and 27.3 %, respectively. Virulence of
R.
solani
isolate on potato may be
depending on the source of isolates from
lesions or sclerotia, as reported by
Carling and Leiner (1990).
Table 1: Pathogenicity tests of 26 fungal isolates on potatoes under
greenhouse conditions during 2018 growing season.
The tested isolates
Isolate code
Disease severity (%)
Dry rot
Whit mold
R. solani
R1
0
0
R. solani
R2
0
0
R. solani
R3
0
0
R. solani
R4
0
0
R. solani
R5
0
0
R. solani
R6
0
0
R. solani
R7
0
0
R. solani
R8
0
0
R. solani
R9
0
0
R. solani
R10
0
0
R. solani
R11
0
0
S. sclerotiorum
S1
0
44.06
S. sclerotiorum
S2
0
37.86
S. sclerotiorum
S3
0
51.8
S. sclerotiorum
S4
0
64
S. sclerotiorum
S5
0
45.83
S. sclerotiorum
S6
0
32.53
S. sclerotiorum
S7
0
38.63
S. sclerotiorum
S8
0
55.4
Fusarium sp.
F1
32.66
0
Fusarium sp.
F2
48.13
0
Fusarium sp.
F3
30.76
0
Fusarium sp.
F4
27.33
0
Fusarium sp.
F5
22.46
0
Fusarium sp.
F6
36.76
0
Fusarium sp.
F7
28.16
0
Control
0
0
LSD at 5%
3.31
3.09
3.3 Effect of
Trichoderma
spp on the
mycelial growth inhibition of the tested
fungi
in vitro
The efficacy of different bioagents on the
mycelial growth inhibition of the tested
fungi was evaluated to study their
antagonistic effects
in vitro
. The
inhibitory effect of biological control
agents,
Trichoderma
spp.
was shown in
Table (2). Data clearly indicated that all
the tested
Trichoderma
isolates were
Mohamed et al., 2021
36
significantly decreased the mycelial
growth of the three pathogenic fungi on
PDA medium compared with the check.
In this respect,
T. harzianum
(T5),
T.
asperellum,
(T34), followed by
T.
harzianum
(T10), exhibited the greatest
mycelial growth inhibition of
R. solani.
as reached 78.9%, 77.8% and 73.2%,
respectively.
Trichoderma harzianum
isolates (No. 7 and 8) showed moderate
inhibition against
R. solani
, while
T.
harzianum
(T9) gave the lowest
reduction of the mycelial growth of the
same fungus. Meanwhile,
T. harzianum
isolates
(No. 10 and 7) and
T.
asperellum,
(T34) gave the best effect in
reducing the mycelial growth inhibition,
of
S. scleroturium.
as reached 75.8%,
69.9% and 69.6%, respectively,
Trichoderma harzianum
(No. 5 and 8)
showed moderate inhibition against
S.
sclerotiorum
, while
T. harzianum
(T9)
gave the lowest reduction of the mycelial
growth of
S. sclerotiorum
. On the other
hand,
T. harzianum
isolates (No. 5 and 7)
and
T. asperellum,
(T34), gave the
greatest inhibition of the mycelial growth
of
Fusarium
sp. as reached 81.3%,
78.13%, 74.4%, respectively. At the
same time
T. harzianum
isolates (No. 8
and 7) showed moderate growth
inhibition against
Fusarium
sp., while
T.
harzianum
(T9) gave the lowest
reduction of the mycelial inhibition of
Fusarium
sp
.
Similarly, this study
showed that the mycelium growth
inhibition of
R. solani
of 75.8%, 69.9%
and 69.6% by
Trichoderma
isolates,
however, in another study, three different
isolates of
Trichoderma
against soil
borne pathogen
R. solani
was observed
to be 74.4-67.8% (Asad et al
.,
2014).
Whatever, some of the antagonists tested
were not found to be very effective
against the pathogens under
in vitro
observations but they may show better
result under their natural field conditions
as their activities depend on the physico-
of the environment (Burgess and Griffin,
1967)
.
Table 2: Effect of different isolates of Trichoderma harzianum sp and T.
asperellum on mycelial growth inhibition of the tested fungi in vitro.
Bioagents
Mycelial growth inhibition (%)
R. solani
S. sclerotiorum
Fusarium sp.
Trichoderma harzianum (T5)
78.96
65.80
81.30
Trichoderma harzianum (T7)
67.36
69.96
78.13
Trichoderma harzianum (T8)
63.53
56.30
73.0
Trichoderma harzianum (T9)
57.86
48.90
69.76
Trichoderma harzianum (T10)
73.20
75.83
70.03
Trichoderma asperellum (T34)
77.86
69.60
74.46
Control
0
0
0
LSD at 5%
3.64
3.84
3.91
3.4 Effect of certain bacterial bioagents
on the mycelial growth inhibition of
the tested fungi
in vitro
Different bacterial bioagents were
evaluated on the mycelial growth of the
tested fungi to study their antagonistic
effects under Lab. conditions. The
inhibitory effect of bacterial agents
was
obtained in Table (3). Data clearly
indicated that all the tested bacterial
bioagents significantly decreased the
Mohamed et al., 2021
37
mycelial growth inhibition of the three
pathogenic fungi on PDA medium
compared with the check. In this respect,
B. subtilis
(Bs1),
B. megaterium
(Bm2)
,
followed by
B. subtilis
(Bs2), showed
that highest reduction of the mycelial
growth of
R. solani,
as recorded 74.76%,
73.0% and 71.1%, respectively. As mean,
B. megaterium
(Bm1) gave moderate
inhibition against
R. solani
, while
P.
fluorescens
(Pf1) gave the lowest
reduction of the mycelial growth
inhibition of
R. solani
. At the same time,
B. subtilis
(Bs2),
B. megaterium
(Bm1)
and
B. megaterium
(Bm2) gave the best
effect in reducing the mycelial growth of
S. sclerotiorum,
as recorded 53.43%,
52.8% and 51.6%, respectively. As
regard,
B. subtilis
(Bs1) showed
moderate inhibition against
S.
sclerotiorum
, while
Penibacillus
polymyxa
(Bp) gave the lowest reduction
of the mycelial growth of
S. sclerotiorum
.
It was shown from the same Table That
P. fluorescens
(Pf2),
P. fluorescens
(Pf1)
and
B. subtilis
(Bs2), gave the best
results in inhibition of the mycelial
growth of
Fusarium
sp., as reached
80.83%, 78.26%, 66.30%, respectively.
Meanwhile,
B. subtilis
(Bs1) gave the
same trend in inhibition against
Fusarium
sp., while
Penibacillus
polymyxa
(Bp) gave the lowest effect in
of the mycelial growth of
Fusarium
sp
.
Among different biological approaches,
the use of the microbial
antagonists like
fungi and bacteria offers an effective,
similar study safely and ecofriendly
strategy to control many of soil-borne
pathogens (Gravel et al
.,
2004). The
isolates and strains of
Trichoderma spp
.
and
Bacillus spp
.
have been proven to be
effective in suppressing plant diseases
caused by
Fusarium
spp. (Kahkashan
and Bokhari, 2012; Abdel-Monaim,
2010).
Table 3: Effect of different bacterial bioagents on the mycelial growth
inhibition of the tested fungi in vitro.
Bacterial bioagents
Mycelial growth inhibition (%)
R. solani
S. sclerotoirum
Fusarium sp.
P. fluorescens (Pf 1)
20.23
33.66
78.26
P. fluorescens (Pf 2)
22.83
42.43
80.83
Pb. polymyxa (Bp)
28.23
26.16
32.80
B. megaterium (Bm1)
68.90
52.80
48.83
B. megaterium (Bm2)
73.03
51.63
46.96
B. subtilis (Bs1)
74.76
46.26
53.16
B. subtilis (Bs2)
71.10
53.43
66.30
Control
0
0
0
LSD at 5%
3.87
3.80
3.73
3.5 Effect of microbial bioagents on
incidence of potato diseases under
greenhouse conditions
In pot experiment, the efficacy of using
B. subtilis, B. megaterium, Penibacillus
polymyxa
,
P. fluorescens
,
T. harzianum,
(No. 5, 7 and 10) and
T. asperellum
(T34) on potato diseases in drenched soil
with the previous bioagents and
artificially infested with the tested fungi
was carried out under greenhouse
Mohamed et al., 2021
38
conditions during 2018/2019 and
2019/2020 growing seasons. Data
obtained in Table (4) clearly showed that
the tested biocontrol agents significantly
reduced the disease severity of potato
diseases compared with control.
Trichoderma harzianum
(T5),
T.
asperellum
(T34)
, Penibacillus polymyxa
(Bp)
, P. fluorescens
(Pf 2)
and
T.
harzianum
(T7) exhibited the highest
reduction of disease severity caused by
R.
solani
which recorded 16.1%, 18.5%,
22.8%, 23.4% and 28.2%, respectively,
followed by
T. harzianum
(T10) and
B.
subtilis
(Bs2) 30.8% and 38.3% during
2019 growing season. While.
Trichoderma harzianum
(T5),
T.
asperellum
(T34),
P. fluorescens
(Pf2)
and
T. harzianum
(T7) as recorded
18.53%, 21.26%, 22.96% and 25.86%
respectively. Also,
Penibacillus
polymyxa
(Bp),
T. harzianum
,
(T10) and
B. subtilis
(Bs2) gave 26.33%, 32.93%
and 37.26% disease severity during 2020
growing season. Whatever,
B.
megaterium
(Bm1) recorded the lowest
reduction of disease severity, reached at
44.2% and 42.96% during 2019 and 2020
growing seasons. The same data showed
that,
T. harzianum
(T5),
and
T.
asperellum
(T34), exhibited the greatest
reduction of disease severity caused with
S. sclerotiorum,
as reached 22.9% and
25.4% during 2019 growing season, as
well as reached at 20.60% and 27.83%
during 2020 growing season.
Table 4: Effect of microbial bioagents on incidence of potato diseases under
greenhouse conditions.
Bioagents
Disease severity %
R. solani
S. sclerotiorum
Fusarium sp.
2019
2020
2019
2020
2019
2020
P. fluorescens (Pf2)
23.4
22.96
33.2
31.2
23.26
24.16
B. polymyxa (Bp)
22.8
26.33
35.26
33.1
20.33
24.3
B. megaterium (Bm1)
44.23
42.96
42.7
38.16
36.1
33.667
B. subtilis (Bs2)
38.33
37.26
35.46
31.26
31.46
36.33
T. harzianum (T5)
16.1
18.53
22.93
20.6
9.6
11.1
T. harzianum (T7)
28.26
25.86
35.5
32.33
27.13
25.83
T. hamatum (T10)
30.8
32.93
32.8
28.46
31.3
28.06
T. asperellum (T34)
18.56
21.26
25.43
27.83
13.1
10.26
Control
73.66
72.03
62.4
60.13
44.33
43.36
LSD at 5%
3.68
3.20
3.30
3.52
3.54
3.36
In this regard,
T. harzianum
(T10),
P.fluorescens
(Pf 2),
B. polymyxa
(Bp),
B. subtilis
(Bs2) and
T. harzianum
(T7)
gave moderate effects in disease
reduction, while
B. megaterium
(Bm1)
recorded the lowest reduction, of disease
severity as reached 42.7% and 38.16%
during 2019 and 2020 growing seasons.
On the other hand,
T. harzianum
(T5)
, T.
asperellum
(T34)
,
B. polymyxa
(Bp), and
P. fluorescens
(Pf 2) gave the highest
reduction of disease severity of dry rot
disease caused by
Fusarium
sp
as
reached 9.6%, 13.1%, 20.3% and 23.2%,
during 2019 growing season. As mean
disease severity reached it 11.10%,
10.26%, 24.30% and 24.16% during
2020 growing season. The same trend
was observed by using
T. harzianum
(T7) and
T. harzianum
(T10) and as
recorded 27.1% and 31.3% during 2019
growing season, 25.83% and 28.06%
Mohamed et al., 2021
39
during 2020 growing season. Meanwhile,
B. subtilis
(Bs2) and
B. megaterium
(Bm1) recorded the lowest reduction of
disease severity. Most frequently species
of
Bacillus
,
Pseudomonas
and
Trichoderma
are used for biological
control of fungal pathogens. Among
them, one of the fungal biocontrol agents
used in this study is
Trichoderma
species.
They are common saprophytic fungi
found in almost any soil and rhizospheric
microflora. The reason for choosing
Trichoderma
species as potential
biocontrol agents because of their ability
to reduce the incidence of disease caused
by plant pathogenic fungi (Dubey et al.,
2007).
3.6 Comparative effectiveness of using
plant extracts against potato diseases
3.6.1
In vitro
test
Ethanolic extracts of six plant leaves
parts and cloves with four different
concentrations
i.e.
5, 10, 15 and 20%
prepared from Basil, Blue gum, Garlic,
Henna, Thyme and Ginger were tested to
study their effectiveness on the mycelial
growth inhibition
of the pathogenic fungi
under lab. conditions. The inhibitory
effect of plant extracts on the tested
fungal growth are shown in Table (5).
The decreasing of the mycelial growth
was increased with increasing the extract
concentrations.
Table 5: Effect of different concentrations of six plant extracts on the mycelial growth
inhibition of the pathogenic fungi.
Plant extracts (A)
Concentration (B) (%)
Mycelial growth inhibition (%)
R. solani
S. sclerotiorum
Fusarium sp
Basil
5
0
0
0
10
0
0
0
15
17.167
15.233
18.167
20
22.967
26.333
29.367
Blue gum
5
35.433
28.367
40.333
10
62.367
58.233
64.133
15
93.8
86.133
89.567
20
100
100
100
Garlic
5
20.9
37.233
46.933
10
51
70.967
80.667
15
76.433
100
100
20
100
100
100
Henna
5
54.3
49.5
60.967
10
84.567
77.267
76.167
15
100
92.867
100
20
100
100
100
Thyme
5
0
0
0
10
0
0
35.167
15
33.9
23.167
72.067
20
61.067
54.967
100
Ginger
5
63.467
54.9
82.733
10
86.333
76.967
94.6
15
95.7
88.333
100
20
100
100
100
Control
-
0
0
0
LSD at 5%
A
1.78
2.01
2.07
B
0.98
0.91
0.84
A × B
2.6
2.42
2.2
Mohamed et al., 2021
40
All the tested plant extracts with any
concentration significantly decreased the
mycelial growth of the pathogenic fungi,
except Basil and Thyme at 5 and 10% on
Fusarium
sp. Among the plant extracts
tested, Henna gave the highest value of
mycelial growth inhibition which
recorded (84.5and 100%) at 10 and 15%
concentrations, Ginger (86.3, 95.7 and
100) followed by Blue gum (62.367,
95.7, and 100) and garlic cloves extract
as reached (51, 76.4and 100%) at 10,15
and 20% concentrations respectively.
Basil and thyme revealed that lowest
mycelial growth inhibition of
R. solani
reached (22.9 and 61.0 %) at 20%
concentrations, respectively
.
Garlic
cloves extract gave the highest value of
mycelial growth inhibition (70.9 and
100%) at 10, and 15% concentration,
Henna (77.2, 92.8 and 100) followed by
ginger (76.9, 88.3and 100) and blue gum
(58.2, 86.1, and 100) at 10,15 and 20%
concentrations respectively. Basil and
thyme (26.3 and 54.9) at 20%
concentration showed the lowest
mycelial inhibition of
S. sclerotiorum
,
ginger gave the highest mycelial
inhibition value (94.6 and 100%), garlic
(80.6 and 100%), Henna (76.1 and
100%) at 10, and 15% concentration
respectively followed by blue gum (64.1,
89.5, and 100) and thyme (35.1, 72.0
and100%) at 10,15 and 20%
concentration respectively, Basil (29.3)
at 20% concentration showed the lowest
mycelial inhibition of
Fusarium
sp
.
The
same effect was obtained by using Henna
extract which strongly retarded and
inhibited the fungal growth. These results
are in line with those reported by
Latif et
al
.
(2006), Abd-El Khair et al
.
(2007) and
Salim (2011) who mentioned that plant
extracts had a good potential to control
various fungal diseases and the inhibitory
effect of the plant extracts might be
attributed to the presence of some
antifungal toxicants.
Table 6: Effect of plant extracts on potato diseases incidence under greenhouse conditions during
2019 and 2020 growing seasons.
Plant extracts (A)
Concentration (B) (%)
Disease severity %
R. solani
S. sclerotiorum
Fusarium sp
2019
2020
2019
2020
2019
2020
Basil
15
61.36
58.23
43.16
40.3
38.06
36.3
20
56.16
53.26
40.53
36.7
35.6
32.6
Blue gum
15
55.5
53.4
38.36
42.5
33.43
30.16
20
52.2
50.46
34.46
37.36
30.0
28.63
Garlic
15
27.53
24.96
23.03
20.26
18.1
20.56
20
22.7
23.43
20.46
19.33
16
18.56
Henna
15
23.13
25.43
31.8
28.43
22.8
24.16
20
20.56
24.83
28.13
25.16
20.6
17.46
Thyme
15
46.2
40.3
33.5
35.86
27.93
23.63
20
43.66
38.33
31.06
33.4
25.8
22.6
Ginger
15
24.53
23.13
27.43
25.1
32.76
35.33
20
23.0
20.96
24
20.63
30.43
33.46
Control
73.66
72.03
62.4
60.13
44.33
43.36
LSD at 5%
A
2.64
2.40
1.34
2.54
2.48
1.64
B
1.29
1.09
1.60
1.20
1.48
1.12
A x B
ns
ns
ns
ns
ns
ns
Mohamed et al., 2021
41
3.6.2 Comparative effectiveness of
plant extracts on potato diseases
incidence
Testing the effectiveness of the plant
extracts was conducted to find out the
best plant extract and concentration that
may inhibit the fungal infection caused
by
R. solani
,
S. sclerotiorum
and
Fusarium
sp.
under greenhouse
conditions. Data obtained in Table (6)
demonstrated that all the tested plant
extracts at 15% and 20% significantly
reduced the infected plants of potato Cara
cv. if compared with the check during
2019 and 2020 growing seasons. Henna
followed by garlic cloves and ginger each
at 15 and 20 % concentrations gave
higher effect in reducing the infected
plants caused by
R. solani.
While, thyme,
blue gum and basil gave the lowest effect
in reducing disease severity during 2019
growing season. On the other hand,
ginger followed by garlic cloves and
henna each at 15 and 20 %
concentrations revealed higher effect in
reducing the infected plants caused by
R.
solani
more than, thyme, blue gum and
basil, which showed lower effect in
reducing disease severity during 2020
growing season. Garlic cloves extract
followed by ginger and henna each at 15
and 20 % had higher effect in reducing
the infected plants caused by
S.
sclerotiorum.
While,
thyme at 20%
concentration exhibited moderate effect
in reducing disease severity, while blue
gum and basil were less effective in
reducing disease severity during 2019
and 2020 growing season.
On the other
hand, garlic cloves extract followed by
henna and thyme each at 15 and 20 %
gave higher effect in reducing the
infected plants caused by
Fusarium
sp.,
While,
ginger at 20% concentration gave
moderate effect in reducing disease
severity, while blue gum and basil
showed the lowest effect in reducing
disease severity during 2019 growing
season. At the same time, Henna
followed by garlic cloves and thyme
extract each at 15 and 20 %
concentrations resulted the highest value
in reducing the infected plants caused by
Fusarium
sp. On the contrary, blue gum,
ginger and basil gave the converse effect
in reducing disease severity during 2020
growing season. Although some
researchers who only used the aqueous
extracts in their studies reported
antifungal effectiveness of this form of
extracts against some fungi (Bhardwaj,
2012) but much documents are available
in favor of present findings from
researchers who compared antifungal
properties of chemically derived and
aqueous extracts (Alizadeh Behbahani et
al., 2013; Jat and Agalave, 2013;
Moorthy et al., 2013; Ambikapathy et al.,
2011; Ashraf et al., 2011).
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