screening of metabolites products of fusarium oxysporum
and determination of its antibacterial and antifungal activity
using medicinal plants extract
abeer fauzi al-rubaye1, imad hadi hameed2, sabreen a kamal1
1department of biology, college of science for women, university of babylon, hillah city, iraq,
2biomedical science department, university of babylon, college of nursing, hillah city, iraq
abstract
the aims of this study were screening of the secondary metabolite products and evaluation antimicrobial
activity. bioactives are chemical compounds often referred to as secondary metabolites. twenty one bioactive
compounds were identified in the methanolic extract of fusarium oxysporum. the identification of bioactive
chemical compounds is based on the peak area, retention time molecular weight and molecular formula.
melissa officinalis was very highly active 6.470±0.25 mm. the results of anti-fungal and anti-bacterial
activity produced by fusarium oxysporum showed that the volatile compounds were highly effective to
suppress the growth of aspergillus fumigatus (5.893±0.20) and streptococcus pyogenes (6.001±0.19). based
on the significance of employing bioactive compounds in pharmacy to produce drugs for the treatment of
many diseases, the purification of compounds produced by fusarium oxysporum can be useful.
keywords: antifungal, antibacterial, fusarium oxysporum, gc-ms, secondary metabolites.
corresponding author:
imad hadi hameed
biomedical science department, university of babylon,
college of nursing, hillah city, iraq phone number:
009647716150716 e-mail: imad_dna@yahoo.com
introduction
fusarium oxysporum is a common inhabitant
of soil and produces three types of asexual spores
macroconidia, microconidia and chlamydospores.
infection by fusarium oxysporum f.sp. cubense triggers
the self-defense mechanisms of the host plant causing
the secretion of a gel. this is followed by the formation
of tylose in the vascular vessels which blocks the
movement of water and nutrients to the upper parts of
the plant1-5. the tips of the feeder roots are the initial
sites of infection which then moves on to the rhizome.
the leaves begin to wilt and may buckle at the base of
the petiole. as the disease progresses, younger leaves
are affected, turn yellow and crumple and the whole
canopy begins to consist of dead or dying leaves. f.
oxysporum is primarily spread over short distances by
irrigation water and contaminated farm equipment6-14.
the fungus can also be spread over long distances either
in infected transplants or in soil. although the fungus
can sometimes infect the fruit and contaminate its seed,
the spread of the fungus by way of the seed is very rare.
it is also possible that the spores are spread by wind.
fusarium oxysporum is an asexual fungus that produces
three types of spores: microconidia, macroconidia,
and chlamydospores14-27. microconidia are one or two
celled, are produced by fusarium oxysporum under all
conditions, and produced the most within the infected
plants. the objectives of this study were analysis of the
secondary metabolite products and determination of
antimicrobial activity.
materials and method
interpretation of mass spectrum was conducted
using the database of national institute of standards
and technology (nist, usa). the database consists of
more than 62,000 patterns of known compounds. the
spectrum of the extract was matched with the spectrum
of the known components stored in the nist library.
fusarium oxysporum was isolated and maintained in
potato dextrose agar slants. spores were grown in a liquid
culture of potato dextrose broth (pdb) and incubated
doi number: 10.5958/0976-5506.2018.00243.7
400 indian journal of public health research & development, march 2018, vol. 9, no. 3
at 25?c in a shaker for sixteen days at 150 rpm. the
extraction was performed by adding 50 ml methanol to
150 ml liquid culture in an erlenmeyer flask after the
infiltration of the culture28-35. the mixture was incubated
at 4?c for 10 min and then shook for 10 min at 130 rpm.
metabolites was separated from the liquid culture and
evaporated to dryness with a rotary evaporator at 45?c.
the residue was dissolved in 1 ml methanol, filtered
through a 0.2 ?m syringe filter, and stored at 4?c for 24
h before being used for gc-ms.
determination of antibacterial and antifungal
activity
the test bacterial pathogens were swabbed in
muller hinton agar plates. 90?l of fungal extracts
was loaded on the bored wells. the wells were bored
in 0.5cm in diameter. the plates were incubated at
37c° for 24 hr and examined. after the incubation
the diameter of inhibition zones around the discs was
measured. fusarium oxysporum isolate was suspended
in potato dextrose broth. they were “flood inoculated
onto the surface of potato dextrose agar and then dried.
standard agar well diffusion method was followed. fivemillimeter
diameter wells were cut from the agar using a
sterile cork-borer, and 25 ?l of the plant samples solutions
were delivered into the wells. the plates were incubated
for 48 h at room temperature. antimicrobial activity was
evaluated by measuring the zone of inhibition against
the test microorganisms. methanol was used as solvent
control. amphotericin b and fluconazole were used as
reference antifungal agent36-47. the tests were carried out
in triplicate. the antifungal activity was evaluated by
measuring the inhibition-zone diameter observed after
48 h of incubation. results of the study were based on
analysis of variance (anova) using statistica software.
a significance level of 0.05 was used for all statistical
tests.
table 1. major phytochemical compounds identified in methanolic extract of fusarium oxysporum.
molecular
phytochemical compound rt (min) weight
serial
no.
1. 1,2,3,4-cyclopentanetetrol , (1?,2?,3?,4?)- 3.150 134.057909
2. 2-furanmethanol 3.259 98.0367794
3. 2,4,6-cycloheptatrien-1-one , 4-methyl- 3.751 120.0575147
4. dihydroxyacetone 3.917 90.031694
5. 2,4-dihydroxy-2,5-dimethyl-3(2h)-furan-3-one 3.968 144.042258
6. 2,3,5-trioxabicyclo[2.1.0]pentane , 1,4-bis(phenylm 3.779 254.094295
7. dl-arabinose 4.134 150.052823
8. isosorbide dinitrate 4.878 236.028066
9. 5- hydroxymethylfurfural 6.738 126.031694
10. 6-acetyl-?-d-mannose 6.686 222.073953
11. l-glucose 7.756 180.063388
12. ?-d-glucopyranoside , o-?-d-glucopyranosyl-(1.fw 10.113 504.169035
13. n-(2,5-dicyano-3,4-dihydro-2h-pyrrol-2-yl)-acetamide 10.960 176.069811
14. 8-hydroxy-2,6-dimethylocta-2,6-dienoic acid ,ethyl 11.458 212.141245
15. 2-acetylamino-3-hydroxy-propionic acid 11.727 147.053158
16. 5h-cyclohepta-1,4-dioxin , 2,3,4a,6,7,9a-hexahydro 14.308 154.09938
17. 7-hydroxy-6-methyl-oct-3-enoic acid 14.525 172.109944
18. trans-2-undecenoic acid 14.662 184.14633
19. 2-heptanol , 6-methyl- 15.979 130.135765
20. dodecane , 1-fluoro- 16.362 188.194029
21. ethylene , 1-nitro-2-[3-benzyloxyphenyl]- 16.608 255.089543
indian journal of public health research & development, march 2018, vol. 9, no. 3 401
table 2. zone of inhibition (mm) of test different bioactive compounds and standard antibiotics of
medicinal plants to fusarium oxysporum.
plant
inhibition
(mm)
plant
inhibition
(mm)
diplotaxis cespitosa 5.870±0.23 daucus carota 5.853±0.22
cassia angustifolia 5.330±0.23 vitex agnus-castus 5.633±0.24
euphorbia lathyrus 6.011±0.22 cressa cretica 6.006±0.25
rosmarinus oficinalis 5.680±0.24 citrus sinensis 6.070±0.22
citrullus colocynthis 4.000±0.17 ruta graveolens 4.080±0.19
althaea rosea 5.074±0.20 thymus vulgaris 6.007±0.25
coriandrum sativum 6.370±0.25 passiflora caerulea 5.900±0.23
origanum vulgare 5.811±0.24 glycine max 5.767±0.22
urtica dioica 3.925±0.23 brassica oleracea 3.908±0.22
foeniculum vulgare 2.989±0.17 olea europaea 3.000±0.19
ocimum basilicum 5.002±0.24 calendula officinalis 5.087±0.23
achillea millefolia 5.514±0.27 taraxacum officinale 2.008±0.20
medicago sativa 2.982±0.18 borago officinalis 3.544±0.19
celosia argentea 3.261±0.21 sambucus nigra 2.015±0.23
apium graveolens 4.801±0.23 c. morifolium 5.906±0.19
brassica rapa 5.973±0.22 equisetum arvense 6.004±0.24
cichorium endivia 5.610±0.24 portulaca oleracea 6.070±0.24
anethum graveolens 6.006±0.23 malva neglecta 5.227±0.19
plantago major 5.002±0.23 l. angustifolia 2.006±0.17
linum usitatissimum 4.075±0.19 althaea officinalis 5.005±0.18
a. esculentus 5.551±0.24 melissa officinalis 6.470±0.25
malva sylvestris 4.991±0.23 control 0.000
results and discussion
identification of biochemical compounds
analysis of compounds was carried out in
methanolic extract of fusarium oxysporum, shown
in table 1. chromatogram gc-ms analysis of the
methanol extract of fusarium oxysporum showed the
presence of thirty one major peaks and the components
corresponding to the peaks were determined as follows.
clinical pathogens selected for antibacterial activity
namely, staphylococcus aureus, staphylococcus
epidermidis, bacillus subtilis, pseudomonas eurogenosa,
escherichia coli, proteus mirabilis, streptococcus
pyogenes, and klebsiella pneumonia maximum zone
formation against streptococcus pyogenes (6.001±0.19)
mm. methanolic extraction of fusarium oxysporum
showed notable antifungal activities against m. canis,
penicillium expansum, aspergillus flavus, candida
albicans, aspergillus fumigatus, trichoderma viride,
saccharomyces cerevisiae, and aspergillus terreus.
aspergillus fumigatus was very highly active against
fusarium oxysporum (5.893±0.20). in agar well
diffusion method the selected medicinal plants were
effective against fusarium oxysporum table 2. fivemillimeter
diameter wells were cut from the agar using
a sterile cork-borer, and 25 ?l of the samples solutions
(anastatica hierochuntica (crude), cassia angustifolia
(crude), euphorbia lathyrus (crude), rosmarinus
oficinalis (crude), citrullus colocynthis (crude),
althaea rosea (crude), coriandrum sativum (crude),
origanum vulgare (crude), urtica dioica (crude),
foeniculum vulgare (crude), and ocimum basilicum
(crude), achillea millefolia, medicago sativa, celosia
argentea, apium graveolens, brassica rapa, cichorium
402 indian journal of public health research & development, march 2018, vol. 9, no. 3
endivia, anethum graveolens, plantago major, linum
usitatissimum, a. esculentus, malva sylvestris, vitex
agnus-castus, cressa cretica, citrus sinensis, ruta
graveolens, thymus vulgaris, passiflora caerulea,
glycine max, brassica oleracea, olea europaea,
taraxacum officinale, borago officinalis, sambucus
nigra, c. morifolium, equisetum arvense, portulaca
oleracea, portulaca oleracea, malva neglecta, l.
angustifolia, althaea officinalis, and melissa officinalis)
were delivered into the wells. melissa officinalis was
very highly antifungal activity (6.470±0.25) mm.
conclusion
twenty one bioactive chemical constituents have
been identified from methanolic extract of the fusarium
oxysporum by (gc-ms). in vitro antimicrobial
determination of products of fusarium oxysporum
forms a primary platform for further phytochemical and
pharmacological investigation.
financial disclosure: there is no financial
disclosure.
conflict of interest: none to declare.
ethical clearance: all experimental protocols were
approved under the department of biology, college of
science, hillah city, iraq.
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