Introduction
Macrophomina phaseolina (Tassi) Goid. is a necrotrophic fungal pathogen responsible for soil- and
seed-borne diseases in up to 500 economically important host plants including mungbean, mashbean, soybean,
sunflower, sorghum, maize, linseed, chickpea and alfalfa (Pawlowski et al.
2015). At initial stages of infection, symptom is not visible but later on they
can appear as black lesions on plant stem, peduncle and branches which
ultimately invade the vascular bundles and causes root rot, collar rot,
seedling blight and damping off disease in plants (Arora 2017). It forms hard
sclerotia, which can survive for long time in the soil and upon favorable
environmental conditions become primary source of infection. The pathogen
becomes more destructive under dry and humid environmental conditions. As the
disease progresses, it provokes the root system destruction along with
chlorosis, growth losses, withering, and ultimately death of a plant (Ullah et al. 2019).
Many
of the fungicides are in practice for the control of fungal pathogens but no
registered fungicide is available in market for the management of M. phaseolina due to the production of sclerotia. The
fungicides have also hazardous effects on environment that makes their usage
restricted (Kalsoom et al. 2019). In this regard scientists
are making efforts to find out environment friendly and cheap bio-products
derived from plants to control the soil-borne pathogens as they are
non-phytotoxic and an excellent substitute to synthetic fungicides. Plants
contain a large number of secondary metabolites such as tannin, terpenoids,
alkaloids, flavonoids, phenols, volatile oils, glycosides and steroids, which
manifest antifungal properties (Khan et
al. 2019). Recently, various reports on different parts of plant extracts
exhibited strong antifungal potential against plant pathogenic fungi under in vitro conditions. In this regard, the
extracts of Sisymbrium irio,
Senna occidentalis, Azadirachta indica, Kochia indica and Sonchus oleraceous have proved very effective to
control M. phaseolina (Javed
et al. 2018; Munir et al. 2018).
Members
of Chenopodiaceae family such as Chenopodium
ambrosioides, C.
album and C. murale
are known to possess several allelochemicals found very useful in obstructing
the growth of phytopathogens especially M.
phaseolina (Javaid and Amin 2009). C.
quinoa also belongs to this family and is known as a pseudo-cereal. It
gained worldwide importance due to its diverse genetic characteristics and
recently introduce in North America, Asia, Africa and Europe. It is commonly
known as quinoa, became an excellent food crop for humans as an alternate to
wheat because of its high nutritious values. In recent years, its cultivation
has also been started in Pakistan on a large scale due to its remarkable
tolerance to salinity, drought and heat (Hernandez-Ledesma 2019). Therefore,
keeping in view that it is a member of family Chenopodiaceae, it was
hypothesized that C. quinoa may also contain antifungal compounds with
potent efficacies against plant pathogens. Therefore, stem extracts of four
varieties of quinoa were explored for their potential to control M. phaseolina
and the detection of antifungal compounds through GC-MS analysis.
Materials and Methods
Antifungal bioassays
For
the collection of plant stem in appropriate quantity, four quinoa verities
namely V1, V2, V7 and V9 were cultivated in winter 2017 in Lahore. Seeds of the
four varieties were obtained from University of Agriculture, Faisalabad,
Pakistan. Details regarding origin of these varieties are given in Table 1. At
the time of maturity, the stems of each variety were collected, dried and
thoroughly crushed. Methanolic extracts were prepared by macerating 200 g of
crushed stems of each variety in methanol (1 L) and kept for two weeks at room
temperature. Thereafter, the mixture was coarse filtrated by muslin cloth and
the extract was concentrated by recovering the solvents on rotary evaporator at
45°C. Stock solution of 15 mL of each extract was prepared in dimethyl sulphoxide (5 mL) by dissolving 9 g of crude methanolic
extracts with subsequent addition of autoclaved distilled water. Similarly,
control solution was prepared without the addition of plant extract. Five
concentration viz. 1, 2,
3, 4 and 5% were formulated by mixing control and stock solutions in suitable
amounts with four replicates of each as reported by Javaid
et al. (2017). M. phaseolina was procured from
Biofertilizer and Biopesticide Lab, IAGS, Punjab University Lahore.
Five-millimeter diameter mycelial plugs of 7-day-old M. phaseolina
culture were added to each conical
flask and left to stand at 28°C. After 7 days, fungal mats were collected on
filter papers and dried in an electric oven at 70°C for data collection.
The variety named V7 was selected and fractionated with different
solvents with increase in their polarities. For this, 3 kg of shade dried,
powdered plant stem was dipped into 10 L of methanol for 15 days and filtered
through a muslin cloth. After that, the thick gummy extract was suspended in
autoclaved distilled water 200 mL and kept for 4 h. The mixture was
successively fractionated beginning with n-hexane
(5 × 500 mL) followed by chloroform (500 mL), ethyl acetate (500 mL) and n-butanol (500 mL) into a separating
funnel. Among these solvents, chloroform and n-hexane fractions were evaporated to obtain their crude extract. The in vitro
biological activity of n-hexane and
chloroform fraction was assessed against M.
phaseolina. Out of the selected extracts, 1 mL of dimethyl sulphoxide
was added to each 1.2 g of the extract to dissolve in followed by the addition
of malt extract 5 mL in order to prepare the sequential concentrations starting
with 200 mg mL-1 and then it was divided into two aliquots. One
aliquot was used for further serial dilution to make the lower concentrations viz.
100, 50, …, 1.562 mg mL-1 and the other one
was used to evaluate extract bio-efficacy. A control was also prepared
similarly in a series without extract addition to maintain the amount of
dimethyl sulphoxide. Inoculum of M. phaseolina was prepared from 8-day-old
culture in autoclaved distilled water. The assay was performed by adding 50 µL
aliquots of the inoculum in each test tube and left to stand at 28°C for 7
days. The obtained fungal mats were filtered and weighed after seven days of
incubation (Shafique et al. 2016).
Three replicates of each treatment were run simultaneously.
GC-MS analysis
GC-MS
analysis of n-hexane and chloroform
fractions was carried out for compounds identification. Ten milligrams of each
of the two fractions were dissolved in 1 mL of their respective solvents and
filtered through Whatman® glass microfiber filters grade GF/C. Analysis was
performed by using a Shimadzu GC-2010plus system coupled with an auto sampler
AOC-20s, an auto injector AOC-20i, and a gas chromatograph. Using helium as a
carrier gas, a volume of 1.0 µL sample was injected by setting injector
temperature at 250°C. The interface temperature was adjusted at 320°C. After
injection of sample, the initial column temperature was 100°C for 60 s that was
enhanced from 100 to 200°C at 20°C min-1 and hold for 2.0 min,
finally from 200°C to 300°C at 40°C min-1. The total run time was
10.9 min.
Statistical analysis
Completely
randomized design was selected for both the laboratory experiments and all the
data were analyzed by ANOVA and LSD test (P≤0.05) using Statistix 8.1.
Results
Antifungal activity
ANOVA
presented in Table 2 indicates that the effect of extract concentration (C),
quinoa varieties (V) and V×C was found to be very effective (P≤0.001) for
the production of fungal biomass. Among the quinoa varieties, V7 methanolic
extract showed a remarkable antifungal activity causing 80–89% suppression of
fungal biomass. V9 extract was ranked as the second most effective antifungal
source against M. phaseolina
where it reduced its growth by 69–88% over control by using different
concentrations. Although methanolic leaf extracts of other two varieties
significantly declined fungal growth but their antifungal potentials were less
pronounced than V7 and V9 as extracts of V1 and V2 inhibited fungal growth by
45–71% and 45–80%, respectively (Fig. 1).
Table 1: Details of four varieties used in
the present study
Quinoa
lines |
Origin |
Plant
name |
V1 |
Colorado, USA |
Colorado 407D |
V2 |
New Mexico, USA |
IESP |
V7 |
New Mexico, USA |
2WANT |
V9 |
Chile |
Pichaman |
Fig. 1: Effect of different
concentrations of methanolic stem extract of four
varieties of C. quinoa 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 LSD test.
Fig. 2:
Effect of different concentrations of n-hexane, chloroform and ethyl acetate fractions of methanolic stem extract of C. quinoa 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 LSD test.
Table 2: Analysis of variance (ANOVA) for
the effect of different concentrations of methanolic
stem extracts of four varieties of C.
quinoa on biomass of M. phaseolina
Sources of variation |
df |
SS |
MS |
F values |
Varieties (V) |
3 |
143547 |
47849 |
328* |
Concentration (C) |
5 |
1305852 |
261170 |
1790* |
V ´ C |
15 |
47138 |
3143 |
21.5* |
Error |
72 |
10505 |
146 |
|
Total |
95 |
1507042 |
|
|
*, Significant at P≤0.001
The highest activity was shown by V7 methanolic extract and was thus
selected for further studies. Different fractions of the extract were effective
against M. phaseolina
(Fig. 2). The selected non-polar fraction n-hexane, and less polar fraction chloroform completely arrested
the growth of fungal pathogen with 1.562 mg mL-1 the lowest MIC
value (Fig. 2A–B). In comparison to the others, two more polar fractions viz. n-butanol
and ethyl acetate were relatively less inhibitory in nature with MIC values of
25 and 12.5 mg mL-1, respectively. There was 52–100% and 66–100%
reduction in M. phaseolina
biomass over control due to the n-butanol
and ethyl acetate fractions (Fig. 2C–D). The aqueous fraction with the highest
polarity showed the least antifungal efficacy by suppressing 46–100% fungal
growth (Fig. 2E).
GC-MS analysis
GC-MS chromatogram of n-hexane indicates the presence of 15
constituents as given in Fig. 3A. The most prevailing compounds were 9,12-octadecadien-1-ol, (Z,Z)- (22.23%) followed by 9,12-octadecadienoic
acid-(Z,Z)-, methyl ester (16.84%) and 1-(+)-ascorbic acid 2,6-dihexadecanoate
(15.18%). Moderately abundant compounds were hexadecanoic
acid, 2-hydroxy-1-(hydroxymethyl)-ethyl ester (10.99%) and hexadecenoic-acid,
methyl ester (7.37%). Whereas, the least abundant compounds
were 1,2-benzedicarboxylic acid,
diisooctyl-ester, ar-tumerone, 6-hexadecenoic-acid, 7-methyl, methyl ester (Z), octadecanoic acid, phytol,
tetradecanoic acid,
curlone, 2-propenoic-acid,3-[4-(acetyloxy)-3-methoxyphenyl]-,
methyl ester, octadecanoic acid, methyl ester and
benzoic-acid,4-hydroxy-3,5-dimethoxy-, hydrazide with peak areas ranging from 4.62 to 1.18% (Table 3; Fig. 4).
Table 3: List of compounds in n-hexane fraction of methanolic stem
extract of C. quinoa identified by
GC-MS analysis
Names
of compounds |
Molecular formula |
Molecular weight |
Retention
time (min) |
Peak
area (%) |
Ar-tumerone |
C15H20O |
216 |
5.797 |
3.75 |
Curlone |
C15H22O |
218 |
5.996 |
2.50 |
Tetradecanoic Acid |
C14H28O2 |
228 |
6.177 |
2.53 |
Benzoic acid,4-hydroxy-3,5-dimethoxy-,hydrazide |
C9H12N2O4 |
212 |
6.258 |
1.18 |
2-Propenoic acid,3-[4-(acetyloxy)-3-methoxyphenyl]-,methyl
ester |
C13H14O5 |
250 |
6.608 |
2.07 |
Hexadecanoic acid, methyl ester |
C17H34O2 |
270 |
6.911 |
7.37 |
1-(+)-Ascorbic acid 2,6-dihexadecanoate |
C38H68O8 |
652 |
7.104 |
15.18 |
9,12-Octadecadienoic acid(z,z)-,methyl
ester |
C19H34O2 |
294 |
7.637 |
16.84 |
Phytol |
C20H40O |
296 |
7.698 |
2.97 |
Octadecanoic acid, methyl ester |
C19H38O2 |
298 |
7.735 |
1.54 |
9,12-Octadecadien-1-ol,(z,z)- |
C18H34O |
266 |
7.837 |
22.23 |
Octadecanoic
acid |
C18H36O2 |
284 |
7.908 |
3.05 |
6-Hexadecenoic
acid,7-methyl,methyl ester (z) |
C18H34O2 |
282 |
8.492 |
3.18 |
Hexadecanoic acid,2-hydroxy-1-(hydroxymethyl) ethyl ester |
C19H38O4 |
330 |
9.645 |
10.99 |
1,2-Benzedicarboxylic
acid, diisooctyl ester |
C24H38O4 |
390 |
9.753 |
4.62 |
Table 4: List of compounds in chloroform fraction of methanolic stem extract of C. quinoa identified by GC-MS analysis
Names
of compounds |
Molecular formula |
Molecular weight |
Retention
time (min) |
Peak
area (%) |
Benzene, nitro- |
C6H5NO2 |
123 |
2.861 |
1.23 |
3-Acetoxy-3-hydroxypropionic acid,methyl
ester |
C6H10O5 |
162 |
2.918 |
1.94 |
4-((1E)-3-Hydroxy-1-Propenyl)-2-methoxyphenol |
C10H12O3 |
180 |
6.168 |
3.28 |
1-Tetracosanol |
C24H50O |
354 |
6.208 |
3.36 |
1-Pentacosanol |
C25H52O |
368 |
6.255 |
6.28 |
2,4-Hexadienedioic
acid,3,4-diethyl-,dimethyl ester,(z,z)- |
C12H18O4 |
226 |
6.436 |
1.81 |
3-Isopropoxy-4-methoxybenzamide |
C11H15NO3 |
209 |
6.538 |
2.01 |
Hexadecanoic acid, methyl ester |
C17H34O2 |
270 |
6.906 |
4.274 |
Piperine |
C17H19NO3 |
285 |
7.020 |
8.43 |
n-Hexadecanoic
acid |
C16H32O2 |
256 |
7.066 |
3.95 |
4,8-Ethano-4H-1,3-benzodioxin,hexahydro- |
C10H16O2 |
168 |
7.142 |
1.99 |
Benzenemethanol,2,5-dimethoxy
acetate |
C11H14O4 |
210 |
7.260 |
6.195 |
Dimethyl
1-(2-methoxyethyl)-5-methylpyrazole-3,4-dicarboxylate |
C11H16N2O5 |
256 |
7.390 |
1.71 |
8,11-Octadecadienoic acid,
methyl ester |
C19H34O2 |
294 |
7.629 |
16.68 |
9,12-Octadecadienoic acid(z,z)- |
C18H32O2 |
280 |
7.795 |
9.14 |
Cis-9-hexadecenal |
C16H30O |
238 |
8.617 |
1.08 |
gamma-Sitosterol |
C29H50O |
414 |
9.343 |
3.49 |
Hexadecanoic acid,2-hydroxy-1-(hydroxymethyl) ethyl ester |
C19H38O4 |
330 |
9.624 |
2.07 |
1,2-Benzedicarboxylic
acid, diisooctyl ester |
C24H38O4 |
390 |
9.752 |
12.48 |
1-Triacontanol |
C30H62O |
438 |
10.335 |
8.60 |
Table 5: Potential antifungal constituents in n-hexane and chloroform fractions of Chenopodium quinoa stem extract
Names
of compounds |
Property |
Reference |
9,12-Octadecadien-1-ol,(Z,Z)- |
Antifungal |
Wang et al. (2008) |
Hexadecanoic acid,2-hydroxy-1-(hydroxymethyl) ethyl ester |
Antifungal |
Al-Marzoqi et al. (2015) |
1,2-Benzedicarboxylic
acid, diisooctyl ester |
Antifungal |
Rahman
and Anwar (2006) |
8,11-Octadecadienoic acid, methyl ester |
Antifungal |
Kianinia and Farjam
(2018) |
2,4-Hexadienedioic
acid,3,4-diethyl-,dimethyl ester,(Z,Z)- |
Antifungal |
Chhouk et al. (2018) |
Chloroform
fraction revealed the presence of 20 compounds (Table 4; Fig. 3B). 8,11-Octadecadienoic acid, methyl ester (16.68%) was present abunduntly followed by 1,2-benzedicarboxylic
acid, diisooctyl ester (12.48%) and 9,12-octadecadienoic_acid_(Z,Z)-
(9.14%). The moderately abundant compounds were 1-triacontanol
(8.60%), piperine (8.43%), 1-pentacosanol (6.28%) and
benzenemethanol,2,5-dimethoxy
acetate (6.195%). However, the least abundant compounds were hexadecanoic_acid, methyl-ester; n-hexadecanoic acid; gamma-sitosterol; 1-tetracosanol;
4-((1E)-3-hydroxy-1-propenyl)-2-methoxyphenol;
hexadecanoic_acid,2-hydroxy-1-(hydroxymethyl) ethyl_ester;
3-isopropoxy-4-methoxybenzamide; 4,8-ethano-4H-1,3-benzodioxin,hexahydro-;
3-acetoxy-3-hydroxypropionic acid, methyl ester; 2,4-hexadienedioic acid, 3,4-diethyl, dimethyl ester
(Z,Z)-; dimethyl
1-(2-methoxyethyl)-5-methylpyrazole-3,4-
Fig. 3: GC-MS chromatograms of n-hexane and chloroform fractions of methanolic stem extract of C. quinoa
Fig. 4: Structures of potential antifungal compounds identified in n-hexane and chloroform fractions of stem extract of C. quinoa through GC-MS
dicarboxylate; benzene,
nitro- and cis-9-hexadecenal with peak
areas ranges from 4.274 to 1.08% (Table
5).
Discussion
In
general, methanolic stem extracts of all the four
quinoa varieties significantly reduced growth of M. phaseolina. Previously, literature
regarding antifungal activity of C.
quinoa is very limited. Woldemichael and Wink
(2001) reported antifungal activity of C.
quinoa against Candida albicans. Saponins,
a diverse group of natural compounds containing steroid aglycone or triterpene
and one or more chains of sugar in their structure (Guçlu-Ustundag
and Mazza 2007), are well known for their antifungal
activity (Tsuzuki et al. 2007). The plant contains at least 26 saponins
(Madl et al. 2006), which might be the reason
of its antifungal activity against M. phseolina (Woldemichael and
Wink 2001). Besides saponins, a
number of other components like eugenol, thymol, carvacrol, phenolics, linalool
and flavonoides are also reported in quinoa that are
known for their antimicrobial properties (Juneja et
al. 2012).
Methanolic extracts showed a marked
variation in different verities towards their antifungal potential. V7
possessed the greatest antifungal potential followed by V9. Similar varietals
differences in antifungal activity have also been recorded among the extracts
of varieties of Vitis vinifera, Allium sativum and Cupressus arizonica against a wide range
of fungal pathogens (Fratianni et al. 2016; Jediyi et al. 2019). Varietals antifungal activity differences could be
attributed to the difference in chemical composition among the varieties (Khouadja et al.
2015). Jediyi et
al. (2019) reported that V. vinifera varieties
were also different in phenols and flavonoids contents so provided a marked
variation in antifungal activities among the selected varieties.
Chloroform and n-hexane
fractions were highly antifungal and completely retarded the growth of the
pathogen even at lower concentrations. To reveal the chemical composition of
these fractions a GC-MS analysis was performed to identify the known antifungal
compounds. Literature survey showed that these compounds might be responsive in
inhibiting the growth of M. phaseolina. Wang et al. (2008) isolated 9,12-octadecadien-1-ol,(Z,Z)- as a major component from Digitaria sanguinalis
and found it to be very effective against Curvularia
eragrostidis. Similarly, Al-Marzoqi et al. (2015)
stated that hexadecenoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester is antifungal in nature against Aspergillus
flavus and A. niger. Likewise,
diisooctyl phthalate also
known as 1,2-benzedicarboxylic_acid, diisooctyl ester was previously isolated from the leaves of Hugonia mystax and Plumbago zeylanica roots as a major
chemical constituent. This
compound was very effective against M.
phaseolina, Alternaria alternata, Botryodiplodia theobromae and Fusarium equiseti
(Rahman and Anwar 2006).
Moreover, a compound namely 8,11-octadecadienoic
acid, methyl ester was also isolated from a medicinal plant Arum maculatum which was found to be effective in arresting
the growth of Penicillium digitatum and A. niger
(Kianinia and Farjam 2018).
Similarly, Chhouk et al. (2018) identified 2,4-hexadienedioic_acid,3,4-diethyl-, dimethyl_ester (Z,Z)-
from Khmer a medicinal plant and reported that this compound possessed
antifungal activity against many pathogentic fungi (Table 5).
Conclusion
There
were large differences among the four selected varieties of quinoa towards
their antifungal potential. M. phaseolina was very susceptible to extracts of V7. The pathogenic growth was completely
controlled when treated with chloroform and n-hexane
fractions of methanic extract of this variety. Various possible antifungal compounds were identified through GC-MS.
Acknowledgements
Seeds
of four varieties of quinoa were provided by Prof. Dr. Shahzad Maqsood Ahmed
Basra, Department of Agronomy, University of Agriculture, Faisalabad
that is highly acknowledged.
Author Contributions
IHK
did experimental work and wrote the paper. AJ supervised the work and
contributed in writing and finalizing the paper.
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