Journal of Phytopathology and Pest Management 6(1): 78-98, 2019
pISSN: 2356-8577 eISSN: 2356-6507
Journal homepage: http://ppmj.net/
∗
Corresponding author:
Waleed M. A. Abd-Elmagid,
E-mail: waleedabdel-magid.5419@azhar.edu.eg
78
Copyright © 2019
Impact of applying certain bio-agents and
plant extracts to control root and pod rot
peanut pathogens
Mohamed M. El-Sheikh Aly
1
, Ali H. ElShaer
2
, Ahmed R. Abdallah
3
, Thrwat A. Aldahtory
3
, Waleed M. A. Abd-Elmagid
1*
1
Agricultural Botany Department, Faculty of Agriculture, Al-Azhar University, 71524 Assiut, Egypt
2
Plant Pathology Research Institute, Agricultural Research Center, Giza, Egypt
3
Agricultural Microbiology Department, Faculty of Agriculture, Minia University, Minia, Egypt
Abstract
Keywords: peanut, plant extracts, Fusarium spp., pod rots, root rot.
The current study was performed to evaluate the efficacy of some bacterial and fungal
bio-agents and certain plant extracts i.e. garlic, neem and mint against Fusarium spp.,
the causal agent of peanut root and pod rot diseases. In vitro tests clarified that
Penibacillus polymyxa (BP) and Pseudomonas fluorecensce (PF2) achieved the highest
inhibition percentage of the tested pathogenic fungi followed by Bacillus subtilis (BS1)
and P. fluorecensce (PF1) while, P. fluorecensce (PF3) and Bacillus megaterium (BM2)
came in the last order. Addition of suspensions of Bacillus (B1), P. fluorecensce (P11),
inoculum of T. harzianum (T10) and combination of all bio-control agents (Mixture) to
infected soil significantly increased yield of peanut plants (Giza 6 cv), such as fresh
weight (gm /plant), dry weight (gm /plant), plant height (cm /plant), number of pods /pot
and weight of pods /pot. Regarding plant extracts, garlic extract was the most effective
plant extract in suppressing the mycelial growth of the pathogenic fungi than any other
treatment. Mint extract showed the lowest effect on reduction of linear growth of the
tested pathogens. Concerning root rot disease management, addition of bio-agents in
vivo resulted in distinct disease reduction obtained by P. fluorecensce (PF2) and Mixture
treatments followed by Penibacillus polymyxa (BP) and T. harzianum (T10), respectively.
In the concern of pod rot disease, the mixture and T. harzianum (T10) treatments
recorded the highest disease reduction as compared to control while, P. fluorecensce
(PF2) and Penibacillus polymyxa (BP) came in the last order. Treated seeds of peanut
(Giza 6 cv.) with certain plant extracts (garlic, neem and mint) at concentration of 30%
significantly reduced the severity of root and pod rot diseases compared to control.
Garlic extract gave the highest reduction of root and pod rot severities on peanut plants
followed by neem extract then mint extract, respectively.
El-Sheikh Aly et al., 2019
79
1. Introduction
Arachis hypogaea
L. (Groundnut or
Peanut) is one of the important crops all
over the world. In Egypt it is one of the
major oil seed crops and considered as
one of the most important, export and
edible oil crops as well as in many
countries of the world. In addition to this,
belonging to leguminous plants, peanut
has the ability to fix nitrogen from the
atmosphere biologically into the soil
which enriches the soil and these benefits
the succeeding crop. Peanut seeds are
characterized by their high oil content
(50%), which is utilized in different
industries beside containing 26–28%
protein, 20% carbohydrates and 5% fiber
(Fageria et al., 1997).
The peanut crop is
vulnerable to soil borne diseases since
both roots and pods of the plant grow in
soil. Diseases caused by soil borne fungal
pathogens reduce yields and the quality
of the harvested pods and it affect the
crop plant until harvest. Pathogens attack
all plant parts of groundnut and restrict
plant development throughout the
growing season as well as reducing seed
quality in post-harvest storage
(Mahmoud
et al., 2006).
Root and pod rot diseases of
peanut are serious worldwide diseases
attacking roots and fruits underground
(Hilal et al., 1990).
Fusarium
spp is
known as a pathogen causing different
symptoms of infected roots and pods
(Hussin Zeinab, 2011; Marei, 2000)
.
Fusarium solani
is one of the most
important pathogens causing brown root
rot in peanuts. Under conductive
conditions like drought stress, the losses
could reach 95% of production in some
fields. Fungicide treatments are not
effective enough to control the disease
beside the harmful effect of excessive and
misuse of fungicides which lead to
environmental pollution and causes
damage to the ecosystem. Recently many
researches tried to develop alternative
control strategies in order to reduce
dependency on synthetic fungicides.
Pseudomonas fluorescence
is an effective
candidate for biological control of soil
borne plant pathogens owing to its
versatile nature, rhizosphere competence
and multiple modes of action beside
being endophytic in the plant system
(Diby et al., 2001).
Pseudomonas
spp.
also can induce systemic biochemical and
ultra-structural changes in the roots that
lead to a greater ability of the host plant
to defend itself against root infecting
pathogens (Sarma et al
.,
2000). It was
noticed that in greenhouse experiments
P. flurescens
and
B. subtilis
significantly
reduced the incidence of all types of
peanut pod rot caused by
R. solani,
S.rolfsii, M. phaseolina, Fusarium
spp.
and
Aspergillus
spp. (Mahmoud, 2004).
Trichoderma
spp. are one of the most
important biological control agents and
one of the most frequently isolated soil
fungi present in plant root ecosystems.
They colonize the root and rhizosphere of
plant and suppress plant pathogens by
different mechanisms, such as
competition, mycoparasitism, antibiosis
and induced systemic resistance,
improvement of the plant health by
promoting plant growth, and stimulation
of root growth
(Mohidden et al., 2010).
Trichoderma harzianum
,
T. hamatum
and
B. subtilis
reduced the mycelial growth of
R. solani, F. solani
and
M. phaseolina
.
Trichoderma hamatum
mainly grew over
the mycelium of the tested pathogens (El-
Sayed et al
.,
2009). It is well known that
plants have the ability to synthesize
aromatic secondary metabolites, like
phenols, phenolic acids, quinones,
flavones, flavonoids, flavonols, tannins
and coumarins (Cowan, 1999).
Therefore, many scientists have reported
the use of plant extracts for controlling
certain fungal diseases (Touba et al
.,
2012; Al-Askar & Rashad, 2010). The
objective of this research was the
El-Sheikh Aly et al., 2019
80
development of alternative control
strategies to reduce dependency on
synthetic fungicides. Also to evaluate
Bacillus
spp.,
Psedomonas florescence
and
Trichoderma
species as potential
biocontrol agents to reduce the impact of
the disease under greenhouse conditions.
2. Materials and methods
2.1 Seeds
Seeds of peanut that have been used in
this study were cultivar Giza 6. They
were obtained from Agriculture Research
Center (ARC), Giza, Egypt.
2.2 Pathogenic fungi source
Virulent
Fusarium
isolates used in this
study were previously isolated, identified
and
proved their pathogenic ability to
induce root and pod rot of peanut in
former study (Abdallah et al., 2016).
2.3 Isolation of biocontrol agents
Isolation trials were conducted to isolate
Pseudomonas
and
Bacillus
colonizing
rhizosphere soil of peanut growing in
different location of Minia, Assiut and
Sohag governorates, Egypt. Isolates were
picked up on the suitable media. All
Pseudomonas
and
Bacillus
isolates were
purified by successive streaking on N-
deficient modified king medium and
nutrient agar media,
respectively,
using
the techniques adopted by King et al
.
(1954) for
Pseudomonas
and nutrient
agar medium
(Oxoid Manual 1965) for
Bacillus.
The purified isolates were
maintained on the same media and
confirmed by microscope examination
for Gram stained young cells (18-24
hours old). Then the purified isolates
were stored at 4°C for further studies. As
for
Trichoderma
spp. it was also isolated
from healthy peanut plants rhizosphere
using dilution plates technique according
to Warcup (1955).
Trichoderma
isolates
were identified according to their
morphological characteristics using
identification keys
(Rifai, 1969), also
two identified isolates obtained were
from Assiut University Mycological
Center, Egypt.
2.4 Antagonistic effect of isolated
microorganisms against
Fusarium
isolates
in vitro
2.4.1 Bacterial biocontrol agents
Six bacterial isolates
i.e.
Pseudomonas
(PF1, PF2, PF3),
Bacillus
(BP, BS1,
BM2) were investigated for their effect
against the growth of
Fusarium
isolates
under Lab conditions. Petri dishes
(containing PDA medium) were streaked
with the bacterial growth which obtained
from 24hours old cultures at the
periphery using sterilized needle and left
for 24 hours. One disk of the pathogen
was placed at the center of each plate,
then plates were incubated at 27°C.
Plates without bacterial inoculation were
served as control treatment. Each
treatment contained three replicates.
When growth of the pathogen covered
the plate surface (9.0 cm in diameter) of
control treatment, antagonistic effect was
determined by measuring the free
inhibition zone, then percentage of
mycelial growth inhibition was
calculated according to the formula:
Percentage of mycelial growth inhibition % = [A – B /A] × 100
Where: A = length of hyphal growth of
the control, B = length of hyphal growth
of the treated.
El-Sheikh Aly et al., 2019
81
2.4.2 Fugal biocontrol agents
Petri-dish was divided into equal halves.
The first half was separately inoculated
with standard disc (5 mm) of
Trichoderma
spp.,
previously isolated
from peanut rhizosphere. The second half
was inoculated with an equal disc of each
of
Fusarium spp.
Isolates.
Each
treatment was replicated three times and
inoculated plates with the pathogen
only
were used as control. All Petri-dishes
were incubated at 27 °C and data were
recorded when control treatments cover
the plates. Antagonistic percentage was
calculated according to the following
scale index: 0-4, 0= no antagonism; 1=
slight antagonism; 2= moderate
antagonism; 3= high antagonism and 4=
overgrowth (Hassan, 1992).
2.4.3 Effect of culture filtrate of
antagonistic fungi on growth of
Fusarium
isolates
Trichoderma
harzianum
isolates (No. 2,
8 and 10) were grown on Czapek′s liquid
medium. Each flask containing 100 ml of
the medium was inoculated separately
with 5 mm agar disc obtained from 7
days-old culture
.
Then, the flasks were
incubated at 27°C for 30 days. The
obtained cultures were used to study the
effect of the culture filtrates of
antagonistic fungi on mycelial growth of
F. oxysporum
(I),
F. solani
(V) and
F.
solani
(VII) isolates. The mycelial
growth was eliminated and the filtrate of
each fungus isolate was centrifuged for
60 min at 3000 rpm to separate the fungal
growth (Mohamed et al., 2008). Filtrates
were sterilized by Seitz filter and added
to the medium at the rate of 10, 25 and
50% (v/v), and mixed thoroughly before
solidification at 40-50°C. Petri-dishes
were inoculated with equal discs
Fusarium oxysporum
(I),
Fusarium
solani
(V) and
Fusarium solani
(VII).
Petri dishes received medium only
without culture filtrate were used as
control. Four replicates were used for
each treatment. All treatments were
incubated at 27°C for 7days. Data were
recorded as diameter of linear growth
when the control plates were completely
covered by the fungal mycelium.
Percentage of reduction in growth was
calculated according to the following
formula (Abd El-Khair & Haggag
Wafaa, 2007):
Growth
inhibition
%
=
((Growth in control –
Growth in each treatment) / Growth in control) ×
100
2.5 Effect of aqueous plant extracts on
growth of pathogenic fungi
Aqueous plant extracts were prepared
from cloves of garlic (
Allium sativum
),
leaves of Neem (
Azadirachta indica
) and
leaves of mint (
Mentha
). The plant parts
were washed several times with sterilized
distilled water , cut into small pieces,
then 100 gram of each were macerated in
100 ml distilled water by using mortar,
resulting extract was squeezed twice
through four layers of cheese cloth, then,
centrifuged at (4000 rpm for 9 min ), and
sterilized using Seitz filtrate (Hassan,
2006). Sterilized filtrates of aqueous
plant extracts were kept in dark bottles in
refrigerator until use. Flasks (250 ml)
contained 100 ml of sterilized PDA
medium were melted. After that, plant
extracts were added to the PDA medium
to obtain concentrations 10, 20 and 30%,
mixed and poured in sterilized Petri
dishes (20 ml /plate). Plates were
El-Sheikh Aly et al., 2019
82
inoculated with equal discs (6 mm in
diameter) of
Fusarium
isolates taken
from 4 days old cultures. Four replicates
were used for each treatment. Control
treatment was obtained by culturing the
tested fungi on PDA medium without
addition of aqueous plant extracts. The
inoculated plates were incubated at 27
±2°C until the fungal growth covered the
plate surface of the control treatments.
The percentage of mycelial growth
reduction was calculated using the
following formula:
Percentage of growth reduction
=
[(growth in control –
growth in treatment) /growth in control] × 100
2.6 Greenhouse experiments
2.6.1 Effect of applying biocontrol
agents to control peanut root and pod
rot diseases
Pot experiments were carried out under
greenhouse conditions in Agricultural
Botany Department, Faculty of
Agriculture, Al-Azhar University (Assiut
branch), Assiut, Egypt during 2015 and
2016 growing seasons. It was done to
study the effect of
Penibacillus polymyxa
(BP),
Pseudmonas fluorescens
(PF2) and
Tichoderma harzianum
(T10) on
controlling root and pod rot of peanut.
Each bacterial suspension (1x10
8
cfu/ml)
was prepared by dilution plate assay as
described by Callan et al
.
(1990).
Bacterial cells from agar cultures of each
isolate were inoculated into nutrient broth
(NB) and centrifuged at 3000 rpm for 5
min, the supernatant was discarded and
the precipitate was re-suspended in 100
ml sterilized distilled water. The
suspension was re-centrifuged for 5 min.
and the precipitate was finally suspended
in sterilized distilled water. Bacterial
concentrations were determined
according to their turbidity using
spectrophotometer. Inoculum of
Trichoderma harzianum
was prepared
according to Abdel-Moneem (1996) by
inoculating sterilized conical flasks
(1000 ml) which contain 75 g sorghum
cereal, 25 g clean sand, 2 g sucrose and
200 ml water with equal discs (0.5 cm)
taken from 7 days old cultures grown on
PDA medium at 27°C. The inoculated
flasks were incubated at 27°C for two
weeks. Bacterial isolates and
T.
harzianum
were applied as soil treatment
by adding 100 ml of bacterial
suspensions (10
8
cfu /ml) and
T.
harzianum
at the rate of 2% (w/w) for
each pot. Inocula of the pathogenic
fungal isolates were prepared by growing
them in sterilized conical flasks (1000
ml) containing sand and sorghum
medium, then incubated at 27°C for 21
days. Sterilized pots (30 cm in diameter)
were filled with infested sand clay soil at
the rate 2% (w/w) 7 days before sowing.
Pots were left for one week, watered and
mixed thoroughly to ensure distribution
with the tested fungi. Eight seeds of Giza
6 cv. were sown in each pots and the
biocontrol agents were applied as
mentioned early. Untreated pots were
served as control. Each treatment was
replicated four times. Disease severity of
root rot was recorded after 90 days from
sowing. The arbitrary (0-5) disease index
scale as described by Grunwald et al
.
(2003) and Hussin Zeinab (2011) was
adopted, where: 0= No visible
symptoms, 1= slight hypocotyls lesions,
2= lesions coalescing around epicotyls
and hypocotyls, 3= lesions starting to
spread into the root system with root tips
starting to be infected, 4= epicotyls,
hypocotyls and root system almost
El-Sheikh Aly et al., 2019
83
completely infected and 5= completely
infected root
.
Pod rot severity was
recorded by adopting the scale based on
area of spots covered on pods and percent
disease index (PDI), calculated mean
disease index. Pods were grouped into
six categories described by Wheeler
(1969), Where 0=non disease symptoms
(disease free), 1=spots cover 1-10%, 2=
spots cover 10-30%, 3= spots cover 30-
50%, 4= spots cover 50-70% and 5=
spots cover >70%. The percentage of
disease severity of root rot and the
percentage of pod rot disease were
estimated using the following formula:
Disease severity (%) = ∑ [(n x V)/5 × N)] × 100
Where, n= number of root or pod within
each infection category, V= numerical
values of infection categories, N= total
number of root or pods examined, 5=
constant, the highest numerical value.
At the end of the experiment some
growth parameters such as plant fresh
and dry weights, plant height, and
number of pods and weight of pods were
evaluated.
2.6.2 Effect of aqueous plant extracts
on the incidence of peanut root rot and
pod rot diseases under greenhouse
conditions
This experiment was conducted to study
the effect of aqueous plant extracts of
garlic, neem and mint on the incidence of
root and pod rots caused by
Fusarium
spp. on peanut plants cv. Giza 6, under
greenhouse conditions during 2015 and
2016 growing season. Preparation of
aqueous plant extracts has been done as
mentioned before. Seeds of cv. Giza 6,
were immersed in the highest
concentration (30%) of each extract for
15 minutes. Sterilized pots (30 cm in
diameter) were filled with sterilized sand
clay soil. Infested soil with each tested
pathogen was carried out as previously
mentioned. Four replicates were used for
each treatment. Each replicate contained
four pots (8 seeds/ pot). Untreated seeds
with aqueous plant extracts were used as
control. After 90 days, diseases severity
was recorded as mentioned before. At the
end of the experiment several growth
parameters such as plant fresh and dry
weights, plant height, and number of
pods and weight of pods were estimated.
2.7 Statistical analysis
Comparison of means was performed
using Fisher’s protected least significant
difference (LSD) at p≤0.05 (Gomez &
Gomez, 1984) and the standard error was
calculated using the statistical analysis
software “CoStat 6.4” (CoStat, 2005)
3. Results and Discussion
3.1 Identification of the biocontrol
agents
Bacterial biocontrol agents isolated from
peanut soil rhizosphere were identified
according to their morphological and
biochemical characteristics as: 3 isolates
as
Bacillus
spp., one isolate as
Penibacillus polymyxa
(BP), one isolate
as
Bacillus subtilis
(BS1), one isolate as
Bacillus megaterium
(BM2) and 3
isolates as
Pseudomonas
florescence
(BF1, BF2 and BF3). Fungal biocontrol
agents were also isolated from peanut
soil rhizosphere; eight isolates were
obtained from rhizosphere soil of peanut
plants plus two isolates obtained from
Assiut University Mycological Center
El-Sheikh Aly et al., 2019
84
(AUMC), Egypt. Fungal isolates were
identified as
Trichoderma harzianum
(1,
2, 5, 7
,
8, 9 and 10)
and
Trichoderma
hamatum
(3, 4 and 6) according to their
cultural and microscopical characters as
described by Rifai (1969).
Table 1: Effect of some antagonistic bacteria on the mycelial growth of the tested fungi in vitro.
Mycelial growth inhibition (%)
Code
Bacterial isolates
F. solani (VII)
F. solani (V)
33.6
58.5
BP
7
Bacillus spp.
30
40.7
BS1
7.8
12.3
BM2
18.3
21.2
PF1
Pseudomonas fluorescence
35
41.8
PF2
8.6
14.3
PF3
0
0
Control
2.20
2.01
L.S.D at 5%
3.2 Antagonistic capability of bacterial
bioagents against mycelial growth of
the pathogenic fungi
in vitro
Data in Table (1) and Figure (1) indicated
that the antagonistic isolates of bacteria
were able to inhibit the mycelial growth
of
F. solani
(V),
F. solani
(VII) and
F.
oxysporum
(I) as compared with the
control.
Penibacillus polymyxa
(BP) gave
the greatest reduction of mycelial growth
of
F. solani
(No. V), followed by isolates
Pseudomonas fluorescens
(PF2),
Bacillus
subtilis
(BS1) and
P. fluorescens
(PF1) as
reached 58.5%, 41.8%, 40.7% and 21.2%
respectively.
Bacillus megaterium
(BM2)
showed the lowest mycelial growth
inhibition of
F. solani
(V), followed by
P. fluorescens
(PF3) as recorded 12.3%
and 14.3% respectively.
Pseudomonas
fluorescens
(PF2),
Penibacillus polymyxa
(BP) followed by
B. subtilis
(BS1)
showed the highest degree in suppressing
mycelial growth of
F. solani
(VII), as
reached 35%, 33.6% and 30%
respectively.
P.fluorescens
(PF1) showed
moderate effect of inhibiting the mycelial
growth of
F. solani
(VII) while,
B.
megatirum
(BM2) followed by
P.
fluorescens
(PF3) gave slight effect on
the mycelial growth of the same fungus
as reached 7.8% and 8.6% respectively.
In the same context,
Penibacillus
polymyxa
(BP),
P.fluorescens
(PF1) and
Bacillus subtilis
(BS1) gave the greatest
inhibition of mycelial growth of
F.oxysporium
(I) as recorded 63.8%,
62.2% and 52.3% respectively.
B.megatirum
(BM2) and
P.fluorescens
(PF1) exhibited moderate effect of
inhibition of the same fungus as reached
49.5% and 47.8%, followed by
P.fluorescens
(PF3) as recorded 33%.
These results are similar to those
obtained by El-Mougy
et al.
(2011)
who
examined the influence of the
antagonistic isolates of
B. subtilis
and
P.
fluorescens
and their culture filtrates
against soil-borne root rot pathogens
R.
solani
and
F. solani in vitro
. The tested
antagonists reduced the linear growth of
fungal pathogens. Pandya
et al.
(2009)
evaluated some antagonists (
B. subtilis
,
T. viride
and
T. harzianum
against
mycelial growth of
F. solani in vitro
. All
the antagonists were effective against
F.
solani
and might be very useful as
potential biological control agents.
Also,
El-Sheikh Aly et al., 2019
85
Mahmoud
et al
. (2006) tested seventeen
bacterial isolates
in vitro
for their
antagonistic effect against the pathogens
F. solani
and
M. phaseolina
. The most
effective isolates in reducing the
mycelium growth of pathogenic fungi
were
P. fluorescens
(Pf2) followed by
B.
subtills
(BS1) and
Bacillus
spp. (BP). In
this respect, Abdel-Monaim (2011) tested
certain bio-control agents (
B. subtilis, B.
megaterium, T. viride
and
T. harzianum)
which isolated from chickpea
rhizosphere, against
R. solani, F. solani
and
S. sclerotiorum
.
Bacillus subtilis
,
B.
megaterium
,
T. viride
and
T. harzianum
gave the highest effect against the tested
fungi
in vitro
.
3.3 Effect of
Trichoderma
harzianum
and
T. hamatum
on linear growth of
the pathogenic fungi
3.3.1 Dual culture
Seven isolates of
Trichoderma
harzianum
and three isolates of
T. hamatum
were
tested to study their effect to inhibit
mycelial growth of
F. solani
(V),
F.
solani
(VII) and F.
oxysporum
(I)
in vitro
on Czapek's agar medium. Data in
Figure (2) indicate that all the tested
isolates of both
Trichoderma
species
significantly reduced the mycelial growth
of
F. solani
(V),
F. solani
(VII) and
F.
oxysporum
(I) although exhibited
different degrees of reaction. The
mycelium of
Trichoderma
spp. grew
rapidly over the mycelium of the tested
pathogens and prevented their
development.
Trichoderma harzianum
T2, T8 and T10 were the most effective
in inhibiting the pathogenic fungal
growth, while isolate T9 exhibited the
lowest effect. On the other hand,
T.
hamatum
isolates showed moderate
effects on the pathogenic fungi mycelial
growth.
Trichoderma harzianum
T2, T8
and T10 were selected for further studies.
Such results are in line with those
reported by Ha (2010).
Antagonistic
effect might be due to direct influence of
antagonistic fungi against pathogens
through coiling their hyphae around the
hyphae of the pathogens to prevent their
continued growth (Adekunle et al., 2006)
and/or production of antagonistic
substances which can play an important
role in lyses of cell wall components of
the pathogenic fungi to help the
antagonists to penetrate the host hyphae
and grow on it as a hyper parasite
(Papavizas et al., 1984). This can be
explained in the light of results recorded
by Abd El-Moity (1981),
who stated that
T. harzianum
works through different
mechanisms,
i.e.
production of gliotoxin,
mycoparasitism and growing very fast
and act as barrier between susceptible
plant tissues and virulent pathogens.
Mycoparasitism by
T. harzianum
is a
complex process, involving recognition
of the host, attachment to the mycelium,
coiling round the hyphae, partial
degradation of the cell wall and
penetration of the host mycelium (Seema
and Devaki, 2012).
Scanning electron
microscopic observation of parasitism of
T. harzianum and T. hamatum
on
R.
solani
revealed that the hyphae of
Trichoderma
coil around the host.
T.
harzianum
attached to host mycelium by
forming hooks and
Trichoderma
produces appressoria at the tips of short
branches.