Introduction
Macrophomina phaseolina, a soil-borne fungus, is well known for causing a number of
diseases mostly charcoal rot in more than 500 plant species including
sunflower, chickpea (Lakhran and Ahir
2020), maize (Emayavarman
et al. 2019), soybean (Yasmin et al. 2020), and other
economically important crop plants (Degani et al.
2020). The pathogen is highly destructive under dry and hot conditions (Pickel et al. 2020). The name charcoal rot is
because of production of large number of minute black microsclerotia by the
fungus which give the plant tissues a black appearance (Sarr
et al. 2014). Management strategies of charcoal rot pathogen include
biological and cultural methods as well as application of fungicides to seeds,
however, these methods have provided only limited disease control (Pandey and Basandrai 2020). Moreover, synthetic agro-chemicals
create environmental problems and also pose risks to human health causing
mutagenic and carcinogenic effects (Singh et al. 2009; Westlund et al. 2018). There is need of an alternate
environmentally friendly strategy for management of M. phaseolina and other fungal pathogens.
Recent studies have shown that natural compounds
from plants and other organisms or their derivatives can be used as fungicides
(Li et al. 2018; Akbar et al.
2020). Various studies revealed that crude plant extracts and pure compounds
are effective in controlling M. phaseolina. Methanolic fruit and leaf extracts of Datura metel were
found highly effective against M. phaseolina (Javaid and Saddique 2012). Moreover,
soil amendment with dry materials of D. metel also significantly reduced charcoal rot disease
in mungbean in pot trial (Javaid
and Saddique 2011). Likewise, methanolic and n-hexane extracts of Chenopodium album, C. quinoa, C. murale and C. ambrosioides
showed profound potential in suppressing in vitro growth of M. phaseolina (Javaid and Amin
2009; Khan and Javaid 2020). Flavonoids isolated from Azadirachta indica and
Mangifera indica had significant
effects in arresting mycelial growth of this pathogen (Kanwal et al.
2010, 2011). Recently, Javaid et al. (2017a)
investigated through bioassays guided fractionation that leaf extract of Senna occidentalis possess highly
antifungal constituents for the management of M. phaseolina. Similar effects of
extracts of Sisymbrium irio, Azadirachta indica and Sonchus oleraceous have also been reported against this fungal
pathogen (Javaid et al. 2017b; Munir et al.
2018; Banaras et al. 2020).
Previous studies have shown that extracts of asteraceous weeds such as Cirsium arvense, Sonchus oleraceous
and Eclipta alba were highly antifungal effective
in inhibiting growth of M. phaseolina (Banaras et al. 2015, 2017, 2020).
However, studies regarding the antifungal effects of asteraceous
weeds Ageratum conyzoids
against M. phaseolina
are lacking. This annual tropical weed is common in West Africa as well as
in parts of South America and Asia where it has been used against a number of
diseases (Marks and Nwachuku 1986). In West Africa,
it is used for wound healing, skin diseases, curing malaria, gastrointestinal
pain, measles, headache and eye diseases (Okunade
2002; Ukwe et al. 2010). Keeping in view
antifungal activity of asteraceous weeds and unavailability
of a registered fungicide against M. phaseolina, the present study was undertaken to
investigate for the antifungal activity of extracts of different parts of A. conyzoides against
M. phaseolina and
identification of possible antifungal compounds through GC-MS analysis.
Materials and Methods
Bioassays with methanolic extracts
A. conyzoides plants were collected from
Lahore, Pakistan and its different parts viz.
leaf, stem, root and inflorescence were separated. Hundred grams of each part
were soaked in 1000 mL of 80% methanol for 2 weeks. Thereafter, materials were
passed through muslin cloth followed by filtration through Whatman No. 1 filter
papers. After evaporation of the solvent on a rotary evaporator (Model ROTVAP,
UTECH Products INC. Albani NY, U.S.A.), the final
traces of the solvent were evaporated in an electric oven at 45°C to get 16,
13, 12 and 10 g of leaf, stem, root and inflorescence extracts, respectively
(Akhtar and Javaid 2018).
For antifungal
bioassays, 9 g of each extract were dissolved in 5 mL dimethyl sulfoxide (DMSO)
and sterilized distilled water was added to prepare 15 mL of stock solution.
Likewise, 15 mL of a control solution were made by mixing the same amount of
DMSO in distilled water. Measured quantities of stock and control solutions
were added to 55 mL pre-autoclaved malt extract broth (MEB) to get 60 mL of
growth medium of each concentration that were divided into 4 equal portions to
serve as replicates. There were six concentrations viz., 0, 1, 2, 3, 4 and 5% (w/v). Experiment was carried out in
100-mL conical flasks with 15 mL growth medium in each flask following Javaid et al. (2018). The flasks were inoculated
with 5 mm plugs of M. phaseolina
(isolated from charcoal rot infected mash bean plants) followed by
incubation at 27°C for one week. Thereafter, fungal biomass was weighed after
filtering and drying at 70°C.
Bioassays with sub-fractions of methanolic stem extract
Methanolic stem extract was
selected for further experimentation on the basis of its best antifungal
activity in laboratory bioassays. Methanolic extract was obtained by soaking 3
kg of crushed stem of the weed for 2 weeks, filtration and evaporation on a
rotary evaporator. To this extract, 200 mL of distilled water was added. It was
partitioned with n-hexane in a
separating funnel. After repeating the process several times for complete
separation of n-hexane soluble
components, the remaining aqueous phase was serially partitioned with 500 mL of
each of chloroform, ethyl acetate and n-butanol.
All the solvents were evaporated on a rotary evaporator and the obtained
sub-fractions were used in antifungal bioassays against the target fungal
pathogen (Javaid et al. 2017b).
Antifungal bioassays
were carried out in 10-mL volume test tubes. A stock solution of 200 mg mL-1
was prepared by dissolving 1.2 g of each sub-fraction in 1 mL DMSO
followed by addition of autoclaved malt extract broth to raise the volume up to
6 mL. Three milliliters of this growth media were
used in antifungal bioassays (1 mL in each test tube) while rest of the volume
was serially double diluted to prepare 100, 50, 25, 12.5, 6.25 and 3.125 mg mL-1
concentrations. For preparation corresponding control treatments, 1 mL of
DMSO was added to 5 mL autoclaved MEB followed by serial double dilutions as in
case of experimental treatments following the procedure given by Javaid et al. (2017b). To each test tube, 20 µL suspension of M. phaseolina was added and tubes were
incubated at 27ºC for one week. Biomass of M.
phaseolina was filtered and weighed after drying
at 70ºC.
GC-MS analysis
GC-MS analysis of chloroform
sub-fraction of methanolic stem extract was performed on Agilant
Technologies Model GC-7890A attached with mass spectrometer MS 5975C.
Statistical analysis
All the data regarding fungal biomass in different laboratory bioassays
were analyzed by ANOVA. Mean separation was carried out by applying LSD test at
P 0.05 using Statistix
8.1.
Results
Bioassays with methanolic extracts
ANOVA
indicated significant differences in plant parts (P), extract concentration (C)
as well as their interaction for biomass production of M. phaseolina (Table 1). Stem extract
exhibited the highest antifungal activity followed by leaf extract causing 20–83%
and 16–67% reduction in biomass of M. phaseolina, respectively. Root and inflorescence extracts showed lower antifungal effects
than the extracts of other two parts of the weeds resulting in 6–31 and 4–21% suppression in fungal biomass over
control, respectively (Fig. 1). In general, fungal biomass was gradually
reduced by increasing the extracts concentrations. A linear association was recorded between fungal
biomass and extract concentration with R2 = 0.9964, 0.9886, 0.9886
and 0.9936 for leaf, stem, root and inflorescence extracts, respectively (Fig.
2).
Table 1: Analysis
of variance (ANOVA) for the effect of different concentrations of methanolic
leaf, stem, root and inflorescence extracts of A. conyzoides on biomass of M. phaseolina
|
Sources of variation |
df |
SS |
MS |
F values |
|
Plant parts (P) |
3 |
44838 |
14946 |
2137* |
|
Concentration (C) |
5 |
73857 |
14772 |
2112* |
|
P ´ C |
15 |
19646
|
1310
|
187* |
|
Error |
72 |
503
|
7 |
|
|
Total |
95 |
138845 |
|
|
*,
Significant at P ≤ 0.001
%20761-767_files/image002.png)
Fig. 1: Effect of methanolic extracts
of different parts of A. conyzoides on biomass of M. phaseolina. Vertical bars show
standard errors of means of four replicates. Values with different letters at
their top show significant difference (P
≤ 0.05) as determined by Tukey’s HSD test
%20761-767_files/image004.png)
%20761-767_files/image006.png)
Fig. 3: Effect of different
concentrations of sub-fractions of methanolic stem extract of A. conyzoides
on growth of M. phaseolina.
Vertical bars show standard errors of means of three replicates. Values with
different letters at their top show significant difference (P ≤ 0.05) as determined by Tukey's
HSD test
%20761-767_files/image008.jpg)
Fig. 4: Percentage decrease in biomass
of M. phaseolina
due to different sub-fractions of methanolic stem extract of A. conyzoides
over control
Fig. 2: Regression analysis for the
effect of different concentrations of methanolic leaf, stem, root and
inflorescence extracts of A. conyzoides on biomass of M. phaseolina
Bioassays with sub-fractions of methanolic stem extract
Generally, higher concentrations
of all the sub-fractions significantly reduced growth of M. phaseolina in terms of its biomass
production. Chloroform fraction showed the highest antifungal potential and all
of its concentrations significantly reduced fungal biomass by 56–93%. The
inhibitory activity was
concentration dependent (Fig. 3B, 4). In n-butanol sub-fraction, although all the
concentrations significantly reduced fungal biomass, however, the effect was less obvious as compared to
chloroform sub-fraction and there was only 24–76% decrease in fungal biomass
(Fig. 3D, 4). In case of n-hexane and
ethyl acetate sub-fractions, the effect of 25–200 mg mL-1
concentrations was significant (P = 0.05).
Different concentrations
of these sub-fractions reduced fungal biomass by 5–70% and 7–75%, respectively
(Fig. 3A and C, 4). The aqueous sub-fraction exhibited the least activity
against M. phaseolina
where only 50% and higher concentrations showed significant effect and different
concentrations reduced fungal biomass just by 4–57% over control (Fig. 3E, and Fig.
4).
GC-MS analysis
GC-MS chromatogram, presented in
(Figs. 5) revealed the presence of 10 compounds in chloroform sub-fraction.
Names and other details regarding retention time, molecular weights and
chemical formulae of the identified compounds are presented in Table 2. Structures of the compounds
are shown in Fig. 6. The
most abundant compound was 2H-1-benzopyran, 7-methoxy-2, 2-dimethyl- (2) followed by hexadecanoic
acid, methyl ester (5);
11-octadecenoic acid, methyl ester (7);
9, 12-octadecanoic acid (Z,Z)-, methyl ester (6) and 1,2-benzenedicarboxylic acid,
mono(2-ethylhexyl) ester (10) with 27.58, 18.85, 15.28, 13.67 and 10.88%
peak areas, respectively. Other compounds namely octadecanoic acid, methyl
ester (8); morphinan,
7,8-didehydro-4,5-epoxy-3,6-dimethoxy-17-methyl-, (5.alpha,
6.alpha)- (9);
1-hexadecanol, 2-methyl- (3); 2-pentadecanone, 6, 10,
15-trimethyl- (4) and
2H-1-benzopyran, 7-methoxy-2, 2-dimethyl- (1) were present in lower concentrations with peak areas ranging
from 2.42 to 3.46%.
Discussion
In the present study, methanolic
stem, leaf, root and inflorescence extracts of A. conyzoides significantly reduced the M. phaseolina fungal biomass. Earlier studies have shown
similar antifungal activities of extracts and essential oils of A. conyzoides against
different fungal species such as Puccinia arachidis
(Yusnawan and Inayati
2018), Fusarium oxysporum (Lian et al.
2019), Penicillium notatum, Rhizopus stolon and Aspergillus niger (Omole et al. 2019). Ethanolic extract of this weed
markedly reduced the growth of F. lateritium, F. solani, Cochliobolus lunatus (Ilondu 2013) and Phytophthora megakarya (Ndacnou et al. 2020). A coumarin compound isolated from acetone fraction of leaves
of A. conyzoides
showed remarkable activity against Aspergilus niger (Widodo et
al. 2012). Likewise, essential
oils of A. conyzoides are known to exhibit
antifungal activity against A. parasiticus and
A. flavus (Nogueira et al. 2010; Patil et al. 2010). Compounds
belonging to flavonoids glycosides, tannins, resins, saponins and
alkaloids are reported to be present in different parts of A. conyzoides (Aja et al. 2016),
most of which are known for their antifungal activity (Kanwal et al.
2010).
Table 2: Compounds identified from
chloroform sub-fraction of methanolic stem extract of A. conyzoides through GC-MS analysis
|
Comp. No. |
Names of compounds |
Molecular formula |
Molecular weight |
Retention time (min) |
Peak Area (%) |
|
1 |
2H-1-Benzopyran, 7-methoxy-2, 2-dimethyl- |
C12H14O2 |
190 |
13.695 |
2.42 |
|
2 |
2H-1-Benzopyran, 6,7-methoxy-2, 2-dimethyl- |
C13H16O3 |
220 |
16.099 |
27.58 |
|
3 |
1-Hexadecanol, 2-methyl- |
C17H36O |
256 |
17.340 |
2.56 |
|
4 |
2-Pentadecanone, 6, 10, 15-trimethyl- |
C18H36O |
268 |
17.926 |
2.53 |
|
5 |
Hexadecanoic acid, methyl ester |
C17H34O2 |
270 |
18.758 |
18.85 |
|
6 |
9,
12-Octadecanoic acid (Z,Z)-, methyl ester |
C19H34O2 |
294 |
20.406 |
13.67 |
|
7 |
11-Octadecenoic
acid, methyl ester |
C19H36O2 |
296 |
20.466 |
15.28 |
|
8 |
Octadecanoic
acid, methyl ester |
C19H38O2 |
298 |
20.653 |
3.46 |
|
9 |
Morphinan,
7,8-didehydro-4,5-epoxy-3,6-dimethoxy-17-methyl-, (5.alpha,
6.alpha)- |
C19H23NO3 |
313 |
23.839 |
2.77 |
|
10 |
1,2-Benzenedicarboxylic
acid, mono(2-ethylhexyl) ester |
C16H22O4 |
278 |
24.246 |
10.88 |
%20761-767_files/image010.png)
Fig. 5: GC-MS chromatogram of
chloroform subfraction of methanolic stem extract of A. conyzoides
Generally, sub-fractions
prepared from methanolic stem extracts of A.
conyzoides retarded the pathogen growth variably
at different concentrations. The variations in antifungal activities of
different sub-fractions of methanolic extracts of other plant species namely Chenopodium album, C. quinoa, C. murale, Coronopus didymus, Senna occidentalis and Sisymbrium irio have also been reported
in other similar studies (Rauf and Javaid 2013; Javaid and Iqbal 2014; Javaid et
al. 2017a, b; Naqvi et al. 2019; Khan and Javaid
2020). This variation may be attributed to different polarity natures of the
organic solvents used for separation of compounds in methanolic stem extract of
A. conyzoides. These
solvents comprised of non-polar n-hexane
on one side and highly polar n-butanol
on the other hand. Compounds present in stem extract were dissolved in various
solvents on the bases of their polarity natures during partitioning process and
thus different sub-fractions showed different antifungal activities. Similar to
that of the present study, in previous studies higher antifungal activities of
chloroform sub-fractions of other plant species have also been reported against
M. phaseolina (Javaid et al. 2017a, b; Khan and Javaid
2020). Recently, %20761-767_files/image012.png)
Fig. 6: Structures of compounds
identified in chloroform sub-fraction of methanolic stem extract of A. conyzoides
through GC-MS analysis
Banaras et al. (2020) have reported similar
antifungal activity of chloroform sub-fraction of an asteraceous
weed S. oleraceous
against M. phaseolina.
Chloroform fraction
was proved to be very effective in suppressing the growth of M. phaseolina in comparison to the other sub-fractions.
Therefore, it was selected for GC-MS analysis that revealed the presence of ten
phytoconstituents, some of which also known for their antifungal activities
against other fungal species. For intense, compound 2 commonly known as precocene (Kouame et
al. 2018), has been identified as one of the major components in essential
oil of A. conyzoides
ranging from 30–93% and inhibited the growth of A. flavus (Castro et al. 2004; Esper et al. 2015).
Iqbal et al. (2004) isolated precocene from A. conyzoides and
reported that 80–100 ppm concentration of this compound can completely control
growth of Sclerotium rolfsii
and Rhizoctonia solani.
Similarly, compound 5, 6, 7 and 8 belong to fatty acid methyl
esters group. Members of this group are generally known for their antifungal
activity against a number of fungal species (Agoramoorthy et al. 2007; Lima et al. 2011; Ali et al. 2017). In the present study,
compound 10 was also found as an
important compound present in reasonable concentration in the chloroform
sub-fraction. This plasticizer compound has been identified in a number of
plants (Polygonum chinense
and Chenopodium album), bacteria (Streptomyces spp.) and fungi (Alternaria spp.), and exhibited
cytotoxic, anti-inflammatory and anti-oxidant properties (Ezhilan
and Neelamegam 2012; Govindappa
et al. 2014; Krishnan et al. 2014; Ali et al. 2017).
Recently, Zhang et al. (2018) have identified this compound in Trichoderma longibrachiatum
showing antifungal activity against a number of phytopathogenic fungi.
Conclusion
The present study concludes that
methanolic stem extract of A. conyzoides and its chloroform sub-fraction are highly
antifungal against M. phaseolina.
The antifungal activity of the chloroform sub-fraction is possibly because
of 2H-1-benzopyran, 7-methoxy-2, 2-dimethyl- as well as 1,2-benzenedicarboxylic
acid, mono(2-ethylhexyl) ester and various fatty acid methyl esters.
Author Contributions
SB
conducted the study, AJ supervised the work and wrote
a part of this paper. IHK contributed in paper writing.
Conflict of Interest
The authors declare no conflict
of interest among them of any sort
Data Availability Declaration
We hereby declare that the data
relevant to this paper is available and will be provided on request
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