Journal of Agricultural Studies
ISSN 2166-0379
2021, Vol. 9, No. 2
Thevetia peruviana (Pers.) K. Schum. Potential
Antifungal Agent Against Mycosphaerella fijiensis
Morelet, Fungi Responsible of Black Leaf Streak
Disease (BLSD) of Plantain (Musa spp)
Jules Patrice Ngoh Dooh (Corresponding author)
Department of Biological Sciences, Faculty of Sciences, University of Maroua, Cameroon,
BP 814, E-mail: ndjuliopat@yahoo.fr
Josué Ngando Essoh
Laboratory of Phytopathology of CARBAP, Njombe, Littoral-Cameroon
Serge Bertrand Mboussi
University of Douala, Laboratory of Plant Biology, PO Box 2701
Alain Heu
Higher Technical Teacher’s Training College, Department of Agriculture and Agropastoral Po
Box 886 Ebolowa
William Norbert Kuate Tueguem
Laboratory of Biotechnologies, Phytopathology and Microbiology Unit, University of
Yaounde I, PO Box, 812, Cameroon
Dieudonne Amayana
Laboratory of Phytopathology, IRAD of Ekona, South west Cameroon
Oscar Nguidjo
Laboratory of Phytopathology of CARBAP, Njombe, Littoral-Cameroon
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Richard Dongmo
Laboratory of Phytopathology of CARBAP, Njombe, Littoral-Cameroon
Kaze Fofe
Laboratory of Biotechnologies, Phytopathology and Microbiology Unit, University of
Yaounde I, PO Box, 812, Cameroon
Paul Martial Tene Tayo
Laboratory of Phytoprotection and Plant Valorization, Biotechnology Center, University of
Yaoundé I P.O. Box 3851, Messa,Yaoundé Cameroon and Department of Biochemistry,
University of Yaound I, Cameroon
Zachee Ambang
Laboratory of Biotechnologies, Phytopathology and Microbiology Unit, University of
Yaounde I, PO Box, 812, Cameroon
Received: March 6, 2021
doi:10.5296/jas.v9i2.18553
Accepted: April 21, 2021
Published: April 22, 2021
URL: https://doi.org/10.5296/jas.v9i2.18553
Abstract
Alternatives to synthetic chemicals are undertaken against phytopathogens. The aim of this
work is to evaluate the effect of seed extracts of Thevetia peruviana (Pers.) K. Schum. on
Mycosphaerella fijiensis Morelet, fungus responsible for banana black leaf streak disease. Five
extracts of T. peruviana, hexane extract (HE), ethyl acetate extract (EAE), acetone extract
(AcE), methanol extract (ME) and aqueous extract (AqE), and a fungicide, Azoxystrobin were
used. GC-MS of acetone extract was performed. Fifty (50) strains of M. fijiensis per sampling
site were tested. Three concentrations of extracts 6.25 (C1), 12.5 (C2), and 25 (C3) μl/ml, a
negative control (0 μl/ml) and 10 ppm of azoxystrobin were used for the tests. The MIC50 and
MIC90 were determined. GC-MS showed chemical compounds with different molecular height
such as acids, sugars, and esters. AcE and AqE significantly reduced M. fijiensis germ tube
growth at C2 and C3 concentrations and with inhibition percentage respectively ranged of
60-90% and 40-80%. The growth levels of the germ tubes were above the strobilurin resistance
threshold at Njombe and peasant plantation, ranging from 77.9% to 92.3%. AcE showed the
same or superior efficacy as the fungicide used on conidial germination at all tested
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concentrations. The MIC50 totally reducing mycelial growth and conidial germination was 6.25
μl/ml. T. peruviana seeds extracts can be exploited in integrated pests management against M.
fijiensis.
Keywords: Thevetia peruviana, Mycosphaerella fijiensis, extracts, GC-MS, Inhibition,
conidia, germ tube
1. Introduction
Fungi are responsible for almpost 60% of crop diseases (Lepoivre, 2003). Their fructifications
are the source of primary inoculum in farms. In Cameroon, Mycosphaerella fijiensis Morelet, a
fungus belonging to Ascomycetes is responsible for the black leaf streak disease (BLSD)of
plantain and sweet banana (Musa sp). Indeed, banana, which is ranked fourth among
agricultural products after rice, wheat and maize, is the most popular fruit on the planet (Lescot,
2006; Lassoudière 2010). This very important sector for Cameroon (first African banana
producer), contributes approximately 7% of primary GDP (Gross Domestic Product) and is the
second largest national employer after the government and the second largest source of income
after timber (Mouliom et al., 1997; Ngando et al., 2006). However, in most banana production
areas, black leaf streak disease is the greatest threat (De Lapeyre et al., 2010). This disease
caused by M. fijiensis affects the photosynthesis of bananas through partial or total drying of
the foliar system of the plant (Mourichon, 2003). This results in yield losses of up to 100%
(Hermento et al., 2010), the reduction of the green lifespan of fruits, making their transport and
conservation problematic (Churchill, 2011).
During dry periods, conidial infection may be a very beneficial form of survival for M.
fijiensis, with the understanding that ascospore infestation is less during these periods
(Jacome and Schuh, 1992). Despite much less conidial production, they can cause disease as
effectively as ascospores (Stover, 1980; Foure and Moreau, 1992; Jones 2009; Ngando et al.,
2015).
More than 30% of the production of banana devoted for export are to the control of losses
due to BLSD over the last 25 years which ranks M. fijiensis first among major agricultural
pathogens (Abadie et al., 1999; Fullerton, 1994; De Lapeyre et al., 2009; Churchill, 2011).
Several control methods are used against this phytopathogen to reduce its effects on crops.
The control of these parasites is accomplished only at the cost of frequent phytosanitary
interventions. Farming practices that aim to reduce inoculum potential in the field by
eliminating necrotic leaves and turning them upside down against the soil (Abadie et al., 1999;
Mourichon, 2003). The development of resistant plants against this disease. However, this
method is considered expensive and very long for farmers. In addition, fungi populations
with a high level of genetic variability are difficult to control, and they can resist any control
measure (El Hadrami, 2000). Some crops like bananas do not have resistant varieties, only
chemical control methods are available for the farmer (Marin et al., 2003). Chemical control
based mainly on the use of synthetic pesticides is the most effective method. This method
unfortunately has consequences on the environment, micro-fauna, micro-flora, and human
health because of the massive and inappropriate use of these fungicides. In addition, it is
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2021, Vol. 9, No. 2
expensive, and it causes the emergence of resistant strains because of the misuse of these
chemical pesticides. This phenomenon of resistance to systemic fungicides has become a
crucial problem for banana plantations in Cameroon (Lepoivre, 2003; Ngando et al., 2006; El
Guilli et al., 2009) and leads to the return to the use of contact fungicides. However, with
contact fungicides, the number of sprays per week increase, which is more expensive.
Awareness of the environmental cost of these practices and consumers' fears of the danger
that pesticide residues accumulated in plant and fish products may pose to human health are
giving rise to a growing interest in other alternatives of control, more efficient and more
environmentally friendly.
Among the alternatives, there is biological control which makes use of biological agents and
their substances (antagonistic microorganisms) on one hand and the use of plant extracts on
the other hand. In the context of integrated pest management, toxic and biodegradable
molecules of plant and microbial origin are essential for reducing the harmful effects of plant
parasites.
Many plants have been reported in the literature to have pesticidal properties against many
plant parasites (Bautista-Banos, 2000; Kassi et al., 2014). The seeds, leaves, fruits and roots
of yellow Oleander (Thevetia peruviana) are considered as potential sources of active
biological compounds for insecticides (Reed et al., 1982; Ambang et al., 2005), rodenticides
(Oji and Okafor, 2000). fungicides (Gata-Goncalves et al., 2003; Ambang et al., 2011; Ngoh
Dooh et al., 2014b; Ngoh Dooh et al., 2015), virucides (Tewtrakul et al., 2002) and
bactericides (Saxena and Jain, 1990). The general objective of this work is to evaluate the
antifungal potential of extracts of Thevetia peruviana against M. fijiensis, agent responsible
for black leaf streak disease.
2. Material and Methods
2.1 Chemical Material
The chemical material consisted of the active ingredient of Bankit, azoxystrobin, obtained
from CARBAP.
2.2 Obtention of M. fijiensis Conidia
Fifty (50) banana plants, spatially representative of the identified plantation area of the
Cavendish AAA variety (dessert banana) in the industrial zone and of the AAB (plantain)
plantain subgroup in the peasant zone (Table I), showing typical symptoms of the disease
Black streak disease were randomly selected from each zone's plantation. Leaf fragments
with stage 2 and stage 3 lesions were collected, wrapped in plastics and transported to the
laboratory. Sampling was done on young leaves of the plant not bearing fruit. Hooks were
used in areas where banana leaves were very high (Ngando et al., 2006, Nguepjop, 2011,
Ngando et al., 2015).
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Table I: sampling sites (PHP: Upper Penja Plantation; CDC: Cameroon Development
Corporation); Peasant: Mbome)
Agro-ecological Region
Plantations
Sector
Peasant
Mbome
Littoral
PHP
Njombe
South West
CDC
Mussaka
2.3 Obtention of the Extracts
The fruits were harvested from different parts of Yaounde City and then crushed with a stone.
The seeds obtained were dried at room temperature in the Phytopathology laboratory of the
Department of Plant Biology of the University of Yaounde I for 3 to 4 weeks. The seeds were
crushed using a hand-mill of brand "Victoria". The resulting powder was loaded into
cartridges and mounted on the soxhlet. The various extraction solvents hexane (HE), ethyl
acetate (EAE), acetone (AcE) and methanol (ME) were each put in turn 48h to 72h (Negrette
et al., 1987). The product obtained was concentrated in a rotary evaporator at the evaporation
temperature of the corresponding solvent to eliminate the latter. The extract obtained was
stored in the refrigerator at 4 °C until use. After this process, the residue was stored and
mounted in the apparatus with another solvent.
The aqueous extract (AqE) was obtained by maceration of the powder in sterile distilled
water for at least 12 hours (Stoll, 1994). The powder was put into a muslin cloth and soaked
in a volume of sterile distilled water necessary to obtain the concentration of the desired stock
solution (36 g in 72 ml of water). The extraction yield of each extract was calculated.
Screening of all these extracts was done previously (Table 2).
Table 2. Occurrence of natural products of each respective extract (Ngoh Dooh et al. 2014a).
Products
Essential oils
Saponifiable oils
Coumarines
Alkaloids
Sterols
Terpenoids
Flavonoids
Anthraquinones
Catechic tannins
Gallic tannins
Saponins
Anthocyanes
Steroic glycosides
Triterpnoid
glycosides
Free sugars
Phenols
Hexane
T
+
_
_
_
_
_
_
_
_
_
_
_
_
Ethyl acetate
+
+
_
+
_
_
_
_
_
_
+
T
_
Acetone
+
+
+
_
+
_
_
_
_
_
+
_
+
_
Methanol
+
_
+
+
+
T
_
_
+
_
+
+
+
T
Aqueous
+
+
+
+
+++
_
+
_
_
+
_
+
_
_
_
_
_
T
_
T
T
+++
_
- Absence of the products, + presence, +++ abundant presence, T presence in traces.
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2.4 Preparation of Concentration of Extracts
A stock solution of 500 μl/ml concentration was prepared by mixing 1ml of the extract, 0.25
ml of solvent and 0.75ml of distilled water giving an initial volume of 2 ml.
From the stock solution, the concentrations of 6.25, 12.5 and 25 μl/ml were obtained by
taking 0.25 ml, 0.5 ml and 1 ml respectively from the stock solution and adding them to
19.75ml, 19.5ml and 19 ml of culture medium, water agar, i.e., a final volume of 20 ml.
(Gata-Gonçalves, 2001). The concentrations of the aqueous extract, 6.25, 12.5 and 25 mg/ml,
were obtained by the same method from a stock solution of 500 mg/ml.
2.5 Dilution of Azoxystrobin
A positive control made of the active ingredient of bankit, a fungicide commonly used against
leaf streak disease, azoxystrobin, was performed. The required dose is that recommended by
the Fungicide Resistance Action Committee (FRAC), International Group of National
Associations of Agrochemical Manufacturers for laboratory tests, or 10 ppm (Essis et al.,
2010; Ngueujop, 2011; Ngando , et al., 2015). A 10000-ppm stock solution was prepared by
mixing 50mg of active ingredient in 5ml of methanol (MeOH) in a tube and all was
homogenized in vortex (VMR). Then 100 µl was taken from the stock solution and added to
99.9 ml of water agar medium.
An antibiotic, Chloramphenicol (200mg/l), was added in the medium to prevent bacterial
contamination of water agar medium.
The volumes obtained were poured aseptically into Petri dishes of 90mm of diameter under a
laminar flow hood the day before the different tests.
2.6 Assessment of the Effects of the Extracts on the Growth of the Germ Tube of M. Fijiensis
A well-insulated lesion, susceptible of bearing conidia on its surface is cut with a scalpel from
each piece of leaf and following its borders to avoid taking several lesions at the same
time.The lower surface of the lesion was applied in the media containingthe different
treatments so as to leave its footprint. Each lesion was first applied to a test control medium
to ensure the effectiveness of the sporulation and then in the treatments. One lesion served as
a source of inoculum for the different treatments and for a concentration in the context of this
monitoring with the conidia method for each site. The Petri dishes were incubated for 48
hours in the culture room at 25 °C and in continuous light.
After 48 hours, observations were made on 50 strains of M. fijiensis for both cultures. The
marks of the lesions in the quadrille and numbered Petri dishes were easily visible under the
microscope. Readings were made following the number of the lesion using a micrometer
(Mouliom et al., 1997; Essis et al., 2010; Nguepjop, 2011).
The first measured criterion is the length (μm) of the inhibited germ tubes which reflects the
action of the extracts and fungicide on medium. The measurement of the length of the germ
tube was done from the last septum or from the insertion base of the conidial germ tube to its
longest end. Three conidia were measured by repetition and the average was selected.
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For each site, and for each concentration, an average length of the germ tubes of 50 conidia
on control (Lc) and on medium amended with extracts or fungicide (Lf) were calculated. The
percentage inhibition of germ tube growth (Ia) was calculated using the formula:
Ia =
Lc − Lf
*100 (Ngando et al.,2006 ; Essis et al., 2010)
Lc
The growth rate (GT) was subsequently evaluated according to the formula GR= 100 - Ia, to
assess the resistance threshold.
The laboratory threshold for reporting resistance was set for Strobilurins (Bankit) at 75% of
control (25% sensitivity) according to FRAC recommendations (Brent and Hollomon, 1998;
Knight et al., 2002; Essis et al., 2010).
2.7 Assessment of the Effects of Extracts on the Germination of Conidia
The different concentrations that were used in the growth test were maintained.
The Petri dishes were incubated in continuous light for 48 h. About 25 to 30 conidia were
counted, some lesions did not 'spit' a lot of conidia. The laboratory threshold for declaring
resistance was set at 80%, i.e., 20% of the sensitivity (Du Pont, 1983; Smith et al., 1991;
Essis et al., 2010).
A spore was considered germinated if the length of the germ tube was equal to or greater than
the diameter of the spore.
The percentages of inhibition were then evaluated according to the following formula:
PI = (A-B) / A X 100
(Leroux et al.,1978 ).
Avec: PI = percentage of inhibition; A = number of spores of germinated in the control
medium; B = number of spores germinated in the presence of the extract or fungicide.
2.8 Acetone Extract Analysis (GC-MS)
The acetone extract which exhibiting the highest percentage of inhibition against M. fijiensis
was analysed by capillary gas chromatography followed by mass spectrometry (GC–MS),
using an Autosystem XL gas chromatograph (Agilent GC 7890A) with a vaporisation injector
in split mode (1:50) interfaced to a Turbomass Perkin-Elmer mass-spectrometer (Agilent
5975 C TAD VL MSD). The analytical parameters were helium as carrier gas with the
column flow rate of 1.21 ml/min. The oven temperature program was 40 ºC for 3 min, then
increased at 5 ºC/min to 180 ºC, followed by 15 ºC/min to 240 ºC and finally to 300 ºC at 10
ºC/min fractions (isothermal 15 min). A fused-silica capillary column, 30×25 mm i.d.30×32
mm (DB-1; 100% di- 150 99. methylpolysiloxane) was used. The ion source and transfer line
temperatures were maintained at 200 and 280 ºC, respectively. Electron ionisation mass
spectra in the range 40–500 Da were recorded at 70 eV electron energy. The scan time was 1
ms, the multiplier potential 430 V and the source pressure 10 Torr. A computer recorded all
data and compounds were identified by comparison with the Wiley’s and Nist libraries
spectral data bank. The fraction previously evaporated and re-suspended in dichloromethane
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was analysed twice (1 ml; ‘‘hot-needle’’) and, for semi-quantitative purposes, the average
percentage composition was computed from peak areas normalised without using correction
factors.
2.9. Evaluation of Minimum Inhibitory Concentrations (MIC50 and MIC90)
From the linear regression equation between the Neperian logarithms of the abscissa
concentrations and the ordinate inhibition percentages, the concentrations reducing growth by
50% (MIC50) and 90 % (MIC90) were determined (Dohou et al., 2004).
Only data from germ tube growth were used to calculate MICs, as recommended by the
FRAC.
2.10 Statistical Analysis
Statistical analysis of the in vitro observation data and study of correlations was done using
the SPSS 16.0 software. The. Principal component analyzes (PCA) were performed using the
XLSTAT 2007.8.04 software to classify the different extracts with respect to azoxystrobin.
The Student Newman Keuls and Duncan tests at the 5% threshold allowed the comparison of
the different averages when the differences were significant.
3. Results
3.1 Acetone Extract Chromatographic and Mains Constituents Found
More than 10 peaks were observed in the chromatographic profil of acetone extract (Figure 1).
Peak retention time varied from 2.71 to 7.19 mn and peak height ranged from 172006 to
1579559.
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Figure 1. GC-MS Chromatographic profil of acetone extract
Many compouds with different molecular weight were obtained from each peak (Table 2).
Acid compounds such decanoid acid, tridecanoid acid and pentadecanoid acid were revealed.
Sugar such as maltose, Ethyl. beta. -d-riboside were obtained. Major compounds were acids
(Table 2). Some compounds were not identified.
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Table 2. Composition of the constituents found in the acetone extract after GC–MS
Peak
(min)
2.71
R.T
Peak height
201922
3.05
254489
3.4
172006
3.78
3889785
4.02
3219504
4.34
509524
4.53
6934187
4.56
5.68
1963294
457213
5.86
1238475
6.32
1579559
Molecular
weight
88
175
117
59
56
72
59
72
56
148
56
103
72
59
54
54
66
68
54
68
68
72
97
74
83
148
166
169
54
128
100
6.42
310154
6.63
4913153
6.65
381820
7.19
1083443
55
66
89
67
80
94
67
80
68
67
109
81
373
Hypothetic compound name
3-Hydroxyphenylacetylene
1-Butanamine, N-methyl-N-nitro2-Butenedioyl dichloride
Ethyl .beta.-d-riboside
Thietane, 2,4-dimethylbeta.-D-Ribopyranoside, methyl
alpha.-D-Glucopyranoside, methyl
Maltose
n-Decanoic acid
1,2-Benzenedicarboxylic acid, bi...
2 Phthalic acid, isobutyl octyl ester
Dibutyl phthalate
Tridecanoic acid
Pentadecanoic acid
cis-13-Octadecenoic acid, methyl
trans-13-Octadecenoic acid, meth...
13-Octadecenoic acid, methyl ester
cis-13-Octadecenoic acid
2 cis-Vaccenic acid
trans-13-Octadecenoic acid
cis-13-Octadecenoic acid
Octadecanoic acid
Glycerol 1-palmitate
Hexadecanoic acid, 2-hydroxy-1Undecanedioic acid
Bis(2-ethylhexyl) phthalate
Phthalic acid, 2-ethylhexyl isoh...
Phthalic acid, cyclohexyl 2-pent
Oleic
acid,
(2,2-dimethyl-1,3-dioxolan-4-yl)
methyl ester
2,3-Difluoroaniline
Hydrazinecarboxylic
acid,
(2-ethoxy-1-methyl-2-oxoethylidene)-,
ethyl ester
None identify.
9,12-Octadecadienal
None identify
9,17-Octadecadienal, (Z)9,12-Octadecadienal
9,12-Octadecadienoyl chloride,
9,17-Octadecadienal, (Z)
9,12-Octadecadienal
cis-13-Octadecenoic acid
9,17-Octadecadienal, (Z)
9,12-Octadecadien-1-ol, (Z,Z)
7,10-Hexadecadienoic acid, methyl
ester
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3.2 Effects of Extracts on the Growth of Germ Tube of M. fijiensis Strains
3.2.1 The Effect of the HE Extract on the Growth of the Germ Tube of M. fijiensis Strains
The HE extract had a very small effect on germ tube growth.No significant difference (P>
0.05)were observed between the differentconcentrationsapplied and in any of the three study.
The highest inhibition percentages recorded were 18.8, 15.0 and 11.5%, respectively at
Njombe (PHP), Mussaka and Mbome at the highest concentration C3. The lowest inhibitions
were obtained in the peasant plantation (1.1 and 1.2% at C1 and C2 concentrations,
respectively. Inhibition of germ tubes by the bankit was highest than that of EH
extract.Highest percentage inhibitions recorded were 50.4, 86.3 and 93.4% respectively in
Mussaka , PHP, peasant zone ((Figure 2).
c
a
a
a
a
b
a
a
a a
Figure. 2. Effect of HE on germ tube growth of M. fijiensis strains
For each site the assigned values of the same letter do not differ significantly according to the
Duncan test at the 5% threshold
T-
C1
C2
C3
T+
T- : 0µl/ml; C1: 6,25µl/ml; C2: 12,5 µl/ml; C3 : 25µl/ml ; T+: 10 ppm
3.2.2. Effect of EAE on germ tube growth of M. fijiensis strains
The EAE extract was shown to be less effective in inhibiting the elongation of the germ tube
of M. fijiensis conidia. An increase in percent inhibition was revealed as a function of
concentration with this extract. Thus, at Mussaka 9.0, 32.8 and 54.6% inhibition, respectively
of concentrations C1, C2 and C3were obtained. The highest sensitivity of the conidia to this
extract was found at the C3 dose with 48.8, 54.6 and 50.8% inhibition, respectively at Njombe
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(PHP), Mussaka and the peasant zone. At Mussaka, this extract was more effective (P <0.05)
than the bankit, at C3 concentration (Fig. 6).
d
d
d
c
c
c
b
b
ab
a
a
Figure 6. Effect of EAE on germ tube growth of M. fijiensis strains
For each site, the assigned values of the same letter do not differ significantly according to
the Duncan test at 5% threshold.
T-
C1
C2
C3
T+
T- : 0µl/ml ; C1: 6,25µl/ml; C2: 12,5 µl/ml; C3 : 25µl/ml ; T+: 10 ppm
3.2.3 Effect of AcE on Germ Tube Growth of M. fijiensis Strains
AcE was very effective in reducing the growth of the germ tube.Inhibition increased with
concentrations in all areas.In the Njombe zone (PHP), 62.1, 84.2 and 95.8% inhibition of
germ tube growth were obtained with C1, C2 and C3concentrations, respectively. In the
peasant zone, inhibition percentages were 62.8, 91.8 and 97.8%, respectively, at the three
concentrations. The AcE extract were more sensitive to germ tube growth compared to the
bankit. At Mussaka, all concentrations showed highest inhibition than azoxystrobin (P<0.05).
In Njombe and Mbome, only, C2 and C3concentrations inhibited germ tube than the bankit.
However, no significant difference was observed between the bankit concentration and the C2
and C3concentrations of AcE at Mbome(P = 0.36), (Fig.7).
All samples were very sensitive to AcE (Fig. 11c) as the lengths of the germ tube were very
short.
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c
b b
b
b b
c c
b
a
a
a
Figure 7. Effect of EAc on germ tube growth of M. fijiensis strains
For each site the assigned values of the same letter do not differ significantly according to the
Duncan test at the 5% threshold
T-
C1
C2
C3
T+
T- : 0µl/ml; C1: 6,25µl/ml; C2: 12,5 µl/ml; C3 : 25µl/ml; T+: 10 ppm
3.2.4 Effect of ME on Germ Tube Growth of M. Fijiensis Strains
Strains from the peasant plantation showed resistance to the methanol extract. The inhibition
percentages were 10.9, 8.2 and 25.1%, respectively at C1, C2 and C3 concentrations. A
sensitivity to the extract was obtained at the C3concentration with samples from the two
industrial zones, 45.8% in Njombe (PHP) and 43.5% in Mussaka (Fig.8). No concentration
matched the effectiveness of azoxystrobin in the areas studied. The lowest inhibition
percentages were obtained with the samples from the peasant zone (P> 0.05).
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c
d
c
d
b
c
a
b
b
aa
a
Figure 8. Effect of ME on germ tube growth of M. fijiensis strains
For each site, the assigned values of the same letter do not differ significantly according to
the Duncan test at the 5% threshold
T-
C1
C2
C3
T+
T- : 0µl/ml; C1: 6,25µl/ml; C2: 12,5 µl/ml; C3 : 25µl/ml ; T+: 10 ppm
3.2.5 Effect of AqE on Germ Tube Growth of M. fijiensis Strains
The aqueous extract proved to be sensitive to the development of the germ tube length at all
concentrations., the germ tubes growth was reduced compared to the control (Fig. 11b). In the
industrial plantation of PHP at Njombe site, the inhibition rates of 40.0, 47.5 and 55.8% were
obtained at different concentration, respectively. The sensitivity was very high with the
samples from peasant plantation, the percentages of inhibition were 81.4 and 75% at C3 and
C1concentrations. At Mussaka, strains were sensitive at C2 and C3 concentrations than Bankit
(P <0.05), the percentages of inhibition were 66.9 and 77.6% against 50.4% for azoxystrobin.
No significant difference (P = 0.114) was obtained at Mussaka between C1 and azoxystrobin
concentrations (Fig 9).
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c
c
c
ba
b
b
a
a
d
a
Figure 9. Effect of AqE on germ tube growth of M. fijiensis strains
T-
C1
C2
C3
T+
For each site the assigned values of the same letter do not differ significantly according to the
Duncan test at the 5% threshold
T-: 0 mg/ml; C1: 6,25 mg/ml; C2: 12,5 mg/ml; C3: 25 mg/ml; T+: 10 ppm
The probability correlation circle of 59.78% revealed four distinct groups. A group consisting
of HE whose inhibition on germ tube growth was very low. The second group containing
EAE, AcE and AqE, which showed an efficiency on germ tube growth. A third group
consisting of ME which showed a moderate efficiency on the inhibition of germ tube growth
of M. fijiensis and finally a group consisting of positive control T + (Fig. 10).
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Figure 10. Mapping of extracts and fungicide effective on inhibition of germ tube growth of
M. fijiensis conidia
a
b
c
1
2
Figure 11. Measurement of the germ tube in the various media supplemented with extracts at
the C3 dose: normal conidia on media, control (a). Conidia very sensitive on media with AqE
(b) and AcE (c). 1 = germ tube; 2 = conidia
3.3 Effect of Different Extracts and Azoxystrobin on the Germination of M. fijiensis Conidia
The HE had no effect on the germination of the conidia of M. fijiensis. The percentages of
inhibition ranged 0-0.5%, 0.2- 0.6% and 0.5-8.6% respectively with samples of PHP,
Mussaka and peasant plantation. No significant difference was obtained between treatment
(P> 0.05%). The lowest value of the percentage inhibition of germination with the bankit was
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obtained in Mussaka, 38.6% against 99.5% in the peasant zone. The germination rates
obtained with this extract werelargely above the resistance threshold of 25% Strobilurins
(Table 3).
The EAE extract was less effective on the germination of M. fijiensis conidia at all
concentrations. With the samples from PHP, the percentages of inhibition obtained were 2.5,
2.5 and 1%, respectively with the doses C1, C2 and C3. At Mussaka the percentages were
ranged 0.2, 2.5 and 3.2%. All inhibition percentages obtained with this extract were lower
than those obtained with azoxystrobin (Table 3).
The AcE proved to be very effective against the germination of M. fijiensis conidia. In
industrial plantations, only the concentration of 6.25 μl/ml gave low inhibition percentages,
8.1% and 38.5% in Njombeand Mussaka, respectively. The C2 and C3concentrations were the
most effective in these industrial zones, with 64.1% and 99% inhibition respectively at
Njombe (PHP) against 85.4% and 96%at Mussaka. The strains of these industrial zones
showed a slight resistance to this extract compared to the samples from the peasant plantation
where the percentages of inhibition were 54.8, 97.8 and 100% respectively at C1, C2 and C3
concentrations. In Njombe (PHP), the C3 dose induced 99% inhibition rate while the
bankitrate was 81.8% (P <0.05). The same result was obtained at Mussaka, the inhibition
percentages obtained were significantly higher and significantly different from those obtained
with the bankit (Table 3). However, in the peasant plantation, no significant difference was
between concentration of extracts and bankit (P = 0.335). Total inhibition was obtained with
samples of peasant zone in the highest concentration, no conidial.
The ME extract was no sensitive to conidial germination. With the samples from the
industrial plantation of Njombe (PHP), the inhibition percentages obtained were 17.8, 11.6
and 34.8%, respectively at the three concentrations. In Mussaka the percentages were 0, 0.6
and 34.6% and 13.4, 7.5 and 9.1% in the peasant plantation respectively at C1, C2 and C3.
Njombe (PHP) strains were slightly more sensitive to this extract than those of the other two
sampling areas. In the three study areas, the bankit showed highest efficacy than this extract
(P <0.05) (Table 3).
AqE was effective against germination of conidia from the peasant plantation and the
Mussaka industrial area. At C2and C3 there were 42.3 and 62% inhibition in Mussaka against
71.5 and 90% in the peasant plantation. Samples from the peasant zone were therefore the
most sensitive to this extract. The synthetic fungicide induced an inhibition of conidial
germination up to 99% in the peasant zone and 38.6% in Mussaka. The bankit was less
effective than the C3 concentration with samples from the Mussaka industrial plantation (P
<0.05). Conidia from the industrial plantation of Njombe (PHP), showed resistance to AqE.
The percentages of inhibition obtained were very low, 10.1, 16.2 and 17.7% respectively at
doses C1, C2 and C3 (Table 3).
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Table 3. Effect of the various extracts and azoxystrobin on the germination of M. fijiensis
conidia
HE
AE
AcE
ME
AqE
T
C1
C2
C3
T+
T
C1
C2
C3
T+
T
C1
C2
C3
T+
T
C1
C2
C3
T+
T
C1
C2
C3
T+
PHP
0.00 ±0
0a±0
0.5 a ± 0.3
0a±0
85b ± 9
0.00 ±0
2.5 a ± 2.2
2.5 a ± 1.3
1 a ± 0.9
85 b ± 9
0.00 ±0
8.1 a ±7.9
64.1 b ± 4.7
99 d ± 4.3
85 c ± 9
0.00 ±0
17.8 a ± 6.5
11.6 a ± 8.3
34.8 b ± 8.1
85 c ± 9
0.00 ±0
10.1 a ± 3.5
16.2 b ± 7.5
17.7 b ± 7.5
85 c ± 9
MUSSAKA
0.00±0
0.2 a ± 0.1
0.1 a ±0
0.6 a ± 0.5
38.6 b ± 7.9
0.00±0
0.2 a ± 0.1
2.5 a ± 1.5
3.2 b ± 1.5
38.6 c ± 7.9
0.00±0
38.5 a ± 7.1
85.4 b ± 3.1
96 c ± 1.4
38.6 a ± 7.9
0,00±0
0 a ± 0.0
0.6 a ± 0.5
34.6 b ± 4.4
38.6 c ± 7.9
0.00±0
9 a ± 4.3
42.3 b ± 3.6
62 c ± 4.6
38.6 ± 7.9
PEASANT
0.00±0
-0.1 a ± 0.0
0.5 b ± 0.4
8.6 b ± 2
99.5 c ± 0.3
0.00±0
7a±1
10 b ± 2.5
24.2 c ± 3.5
99.5 d ± 0.3
0.00±0
54.8 a ± 5.4
97.8 b ± 0.8
100 b ± 0.0
99.5 b ± 0.3
0.00±0
13.4 a ± 5.7
7.5 a ± 3.2
9.1 a ± 3.3
99.5 b ± 0.3
0.00±0
80 b ± 4.3
71.5 a ± 4.9
90 c ± 2.3
99.5 c ± 0.3
For each site and each extract, the values assigned to the same letter do not differ
significantly at the 5% threshold according to the Duncan test.a) T-: 0 mg/ml; b) C1 : 6,25
mg/ml ; c) C2 : 12,5 mg/ml; d) C3 : 25 mg/ml; T+: 10 ppm
The 56.28% probability threshold correlation circle revealed the existence of three distinct
groups in the efficiency of the extracts in the inhibition of M. fijiensis conidia germination. A
group consisting of HE which was ineffective, another group consisting of effective extracts,
AcE, AqE and T+, and a last group for the extracts with low efficiency, ME and EAE (Fig.
12).
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Figure 12. Mapping extracts and fungicide effective on the inhibition of conidial germination
of M. fijiensis
4. Minimal Inhibitory Concentrations MIC50 and MIC90 of germ tube growth of M.
fijiensis strains
The lowest MICs were obtained with AcE and AqE. MIC50 ranges from 2.0 μl/ml to 3.3 μl/ml
for AcE and from 6.0 mg/ml to 15.0 mg/ml for AqE. The highest MICs were obtained with
HE. Thus, with EAE, the MIC50 values varied from 20.1 to 30 μl/ml respectively for Mussaka
and Njombe (PHP) and with the ME from 27.1 to 403.4 μl/ml. However, the MIC50 and
MIC90 of AcE were substantially equal in all the zones (3.3, 2.0 and 3.3 μl/ml) which
indicates the same behavior of the extract with respect to all strains regardless of the
sampling site (Table 4).
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Table 4. CMI50 and CMI90 of the growth of the germ tube of M. fijiensis with the different
extracts tested (µl/ml or mg/ml)
Treatment
Sites
HE
CMI50 CMI90
EAE
AcE
ME
CMI50 CMI90
CMI50 CMI90
CMI50
PHP
*
30,0
167,2
3,3
14,7
*
MUSSAKA
*
20,1
73,4
2,0
18,0
27,1
PEASANT
5431 1,2106
24,5
97,7
3,0
18,0
AqE
CMI90
*
74,7
403,4 1066
CMI50
CMI90
15,0 525,1
6,0
*
40,1
369,2
*values no founded (negative correlations)
5. Discussion
The present study was based on the extraction of natural substances from the seeds of T.
peruviana and on the evaluation of the antifungal potential of its extracts on M. fijiensis,
responsible for the disease of black streaks of banana.
The chromatographic of The AcE has showed many major compounds, its effectiveness could
be due to the presence of molecules that act in high doses. Gata-Goncalves et al. (2003) also
showed after GC-MS of extract of T. peruviana, presence of many compounds
Plant extracts have already shown their effectiveness against the germination of fungal spores.
This is the case with the results of Achraf et al. (2012) which found out that the aqueous
extract of Asphodelus tenuifolius and Zygophyllum albumwere effective in inhibiting spore
germination of Penicillum expansum with a percentage inhibition of 95.48% and 93.82%.
The ethanol extracts of the leaves of O. gratissimum and Aframomum melegueta prevent the
spores of Fusarium oxysporum and Aspergillus niger from germinating by more than 65%,
according to the findings of Okigbo & Ogbonnaya (2006).
The AcE greatly reduced the germ tube elongation of M. fijiensis strains, followed by EAq.
The AcE was very effective in inhibiting the germination ofM. fijiensis strains in all banana
growing areas studied, being more effective than the chemical fungicide azoxystrobin used. It
inhibited more than 90% conidial germination. These results are similar to those of
Arciniegas, (2002) and Arciniegas et al. (2002) who showed an in vitro test a strong
antifungal activity on both colony development and germination of M. fijiensis conidia using
crude ethanol extracts, amphipolar solvent as well as acetone extract of eight plants such as
Commelina diffusa, Momordica charantia, Piper hispidum, Piper peltatum, Sida rhombifolia
and Syzyrgium aromatica.
Furthermore, Paola (2006) obtained a significant reduction in the incidence and severity of
black leaf streak disease on plants infected artificially with M. fijiensis conidia after being
treated with extracts obtained from the maceration of Momordica charantia and Senna
reticulata in water/alcohol mixture. Indeed, the phytochemical analysis performed on S.
reticulata with which they obtained the best results revealed the presence of a variety of
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secondary metabolites such as polyphenols, coumarins, saponins, triterpenes, flavonoids
some of which are found in the extract with acetone (AcE) from T. peruviana
(Gata-Goncalves et al., 2003; Ngoh Dooh et al., 2014). These extracts are known for their
antifungal activity or resistance to the induction on M. fijiensis (Riveros et al., 2003; Polanco,
2004).
The growth and germination rates obtained with HE, EAE and ME were significantly higher
(77.9% to 92.3%) at the reported Strobilurin resistance threshold which shows the resistance
of the strains of these zones to these extracts. Only the C3 doses of EAE and EAq gave lower
rates with Mussaka strains. The growth and germination rates obtained for the EAq at doses
C1, C2 and C3, despite the effectiveness of this extract did not fall below the resistance
threshold of Strobilurins. The growth and germination rates achieved with EAc were below
the Strobilurin resistance level at C2 and C3 on growth and in all areas at C2 and C3 with
Mbome and Mussaka strains and the C3 dose at the PHP, on the germination, which shows the
sensitivity of the strains of these zones to this extract.
With the bankit, the growth level of the germ tube was below the strobilurin resistance
threshold in two of the three study areas (Hermento et al., 2010). The lowest rate was
obtained in the plantation, 7.6%, followed by the Njombe industrial plantation. An onset of
resistance was obtained in Mussaka with azoxystrobin (49.6%).
The bankit whose active ingredient, azoxystrobin, was used in this study was found to be
effective in some production areas compared to others. Azoxystrobin, which belongs to the
Strobilurins class, is a potent inhibitor of cell respiration, hence its effectiveness against germ
tube elongation and germination (both parameters being linked). This result corroborates that
of Essis et al. (2010) who obtained an efficacy in the laboratory of azoxystrobin against the
germ tube growth of M. fijiensis strains from Ivory Coast banana plantations. However, in
Mussaka a resistance to this fungicide was detected. This could be explained by the abusive
use of this compound which eventually developed resistant strains compared to the peasant
zone where there is no use of the bankit. This result is contrary to that of Nguepjop (2011)
which indicated a lack of resistant strains in this zone. Ngando et al. (2006) showed
resistance phenomena in banana plantations in Cameroon. The low efficiency of azoxystrobin
in the peasant plantation denotes the appearance of resistant strains, despite the distance with
the industrial zones. This could be explained by the movement of men from one corner to
another and who would carry conidia resistant strains to areas where they do not exist
(peasant zone).
The MIC50 of the different extracts were determined with the different strains tested. Low
variability was observed. The low values and close to the MICs obtained with the acetone
extract demonstrate the efficacy and fungicidal properties of this extract in inhibiting the
germination of the conidia of the fungus tested. These results are in agreement with those of
Doumbouya et al. (2012) who showed that the low MIC values of Ocimum graticimum
extracts inhibit the development of phytopathogenic fungi.
The inhibition percentages obtained were different according to the extracts. This can be
explain by the process of obtention the extracts. Okigbo (2005) showed that the levels of
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bioactive compounds in plants with antifungal activity could be influenced by many factors,
including the plant’s age, harvest time, extraction solvent, and method of extraction. Indeed,
for the same species of fungi, an extract preceding or following another could show an
effectiveness contrary to its predecessor or its successor. Thus, AcE was effective in
inhibiting the germination of all strains studied, unlike methanol which showed no efficacy
meanwhile these two solvents are polar. The extraction with acetone would have taken the
active ingredient acting on the germination before the application of methanol.
The plant used in this study belongs to the Apocynaceae family. The extracts contain many
chemical compounds. These compounds could act on the target organisms in several ways.
They could inhibit growth by acting on metabolic functions such as cell division. Others
would inhibit respiration by blocking ATP production or inducing plant resistance (Laurent et
al., 2003; Lepoivre 2003; Chwaleka et al., 2006).
6. Conclusion
The AcE and AqE were effective as azoxystrobin, the active ingredient in the bankit, a
fungicide commonly used against growth tube length and germination of conidia of M.
fijiensis responsible of black leaf streak disease. The GC-MS revealed many compounds with
antifungal properties.
Acknowledgement
Our thanks to the Director of CARBAP (Plantain Banana Research Center) of Africa, Dr.
Ngando Essoh and the entire team (technicians) of the CARBAP Phytopathology Laboratory
(Mpouli, Nguidjo, Dongmo) with who the sampling in field was done and Dr. Amayana,
Former Scientific Director of the IRAD of Ekona in South West Cameroon.
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