characterization of antibacterial and antifungal metabolites
produced by macrophomia phaseolus and analysis of its
chemical compounds using gc-ms
abeer fauzi al-rubaye1, ghaidaa jihadi mohammed2, imad hadi hameed3
1 department of biology, college of science for women, university of babylon, hillah city, iraq,
2department of biology, college of science, university of al-qadisiyah, hillah city, iraq,
3biomedical science department, university of babylon, college of nursing, hillah city, iraq
abstract
twenty one bioactive compounds were identified in the methanolic extract of macrophomia phaseolus.
gc-ms analysis of macrophomia phaseolus revealed the existence of the benzonitrile , m-phenethyl,
carbamic acid, n,n-dimethyl-,3,4-dimethylphenyl ester, 3-cyano-6-oxopyrazolo[3,4-d]pyrimidin-4-thione,
4h-benz[b]1,4-oxazin-2(3h)-one, 7-nitro-3-(2-oxo-2-phenylethy, 1-benzenesulfonyl-1h-pyrrole, pentyl
glycolate, ethyl fluoroformate, cyclopropyl-3,4-epoxyhex-5-en, benzimidazole ,7-methyl-, 1,1-difluorocis-
2,3-dimethyl-cyclopropane, pyrazolo[1,5-a]pyridine, 3-methyl-2-phenyl-, n,n-dimethyl-3-methoxy-4-
methylphenethylamine, 1,1’-bicyclohexyl , 4-methoxy-4’-propyl-, 1-benzylindole, and benzenemethanol
,2-chloro-?-[[(1-methylethyl)amino) methyl. datura stramonium (alkaloids) was very highly active
6.61±0.26 mm. the results of anti-fungal and anti-bacterial activity produced by macrophomia phaseolus
showed that the volatile compounds were highly effective to suppress the growth of aspergillus flavus
6.009±0.23mm and proteus mirabilis 6.38±0.22mm.
keywords: antifungal, antibacterial, macrophomia phaseolus, 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
macrophomina phaseolina is a botryosphaeriaceae
plant pathogen fungus that causes damping off, seedling
blight, collar rot, stem rot, charcoal rot, basal stem rot,
and root rot on many plant species1-5. the m. phaseolina
fungus has aggregates of hyphal cells, which form
microsclerotia within the taproots and stems of the
host plants. the pathogen m. phaseolina affects the
fibrovascular system of the roots and basal internodes
of its host, impeding the transport of water and nutrients
to the upper parts of the plant. as a result, progressive
wilting, premature dying, loss of vigor, and reduced yield
are characteristic symptoms of m. phaseolina infection.
the fungus also causes many diseases like damping off,
seedling blight, collar rot, stem rot, charcoal rot, basal
stem rot, and root rot. the hyphae infect the roots of the
host plant. initially, the hyphae enter the cortical tissue and
grow intercellularly, then infect the roots and the vascular
tissue. within the vascular tissue, mycelia and sclerotia
are produced and plug the vessels 6-11. m. phaseolina is
a heat- and drought-favoring disease, producing large
quantities of microsclerotia under relatively low water
potentials and relatively high temperatures. in soybeans
especially, charcoal rot typically occurs when the plants
are experiencing significant drought stress 12, 13. the aims
of this study were analysis of the metabolite products
and determination antimicrobial activity.
materials and method
analysis of bioactive compounds
gc-ms analysis was done on a thermo gas
chromatography mass spectrometer (agilent 789 a)
equipped with db-5 capillary column (30 m long,
doi number: 10.5958/0976-5506.2018.00240.1
382 indian journal of public health research & development, march 2018, vol. 9, no. 3
0.25 mm i.d., film thickness 0.25 ?m). the column
temperature program was 50 °c for 6 min, with 5 °c
increases per min to 250 °c which was maintained for
30 min. macrophomia phaseolus was isolated from dried
fruit and the pure colonies were selected, isolated and
maintained in potato dextrose agar slants 14-19. spores
were grown in a liquid culture of potato dextrose broth
(pdb) and incubated 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 culture. 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 20, 21. 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 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
22-28. after the incubation the diameter of inhibition zones
around the discs was measured. macrophomia phaseolus
isolate was suspended in potato dextrose broth and
diluted to approximately 105 colony forming unit (cfu)
per ml. they were “flood inoculated onto the surface of
potato dextrose agar and then dried. standard agar well
diffusion method was followed 29-33. five-millimeter
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 34-38.
table 1. bioactive chemical compounds identified in methanolic extract of macrophomia phaseolus.
benzonitrile , m-phenethyl-
rt=3.156
mw=207.104799
pharmacological activity: unknown
carbamic acid , n,n-dimethyl-,3,4-
dimethylphenyl ester
rt= 3.161
mw= 193.110279
pharmacological activity: anti-oxidative
potential
3-cyano-6-oxopyrazolo[3,4-d]
pyrimidin-4-thione
rt= 3.264
mw= 193.005831
pharmacological activity: antifungal
activity
4h-benz[b]1,4-oxazin-2(3h)-one ,
7-nitro-3-(2-oxo-2-phenylethy
rt= 3.819
mw= 310.058971
pharmacological activity: unknown
1-benzenesulfonyl-1h-pyrrole
rt= 4.014
mw= 207.035399
pharmacological activity: antimicrobial
activity
pentyl glycolate
rt= 4.077
mw= 146.094295
pharmacological activity: antifungal
activity
ethyl fluoroformate
rt= 4.237
mw= 92.0273576
pharmacologicalactivity:
anti-inflammatory activity
1-cyclopropyl-3,4-epoxyhex-5-en-1-yne
rt= 4.226
mw= 134.073165
pharmacological activity:
anti-.inflammatory, anticancer
benzimidazole ,7-methyl-2-phenyl-
rt= 4.460
mw= 208.100048
pharmacological activity:
antimicrobial, antiviral
1,1-difluoro-cis-2,3-dimethylcyclopropane
rt= 4.397
mw= 106.0594068
pharmacological activity: unknown
pyrazolo[1,5-a]pyridine , 3-methyl-2-phenyl-
rt= 4.649
mw= 208.100048
pharmacological activity: unknown
l-proline , n-(cyclohexanecarbonyl)-
,propyl ester
rt= 4.992
mw= 267.183443
pharmacological activity: unknown
indian journal of public health research & development, march 2018, vol. 9, no. 3 383
n,n-dimethyl-3-methoxy-4-
methylphenethylamine
rt= 5.622
mw= 193.146665
pharmacological activity: antiinflammatory
activity
1,1’-bicyclohexyl , 4-methoxy-4’-propyl-
rt= 7.235
mw= 238.229666
pharmacological activity: anti-.
inflammatory, anticancer
1-benzylindole
rt= 7.882
mw= 207.104799
pharmacological activity: antiinflammatory
3-amino-7-nitro-1,2-benzotriazine
1-oxide
rt= 8.986
mw= 207.039239
pharmacological activity: antiviral,
anticancer, anti-inflammatory
benzenemethanol ,2-chloro-?-[[(1-
methylethyl)amino) methyl]-
rt= 9.850
mw= 213.092042
pharmacological activity:
unknown
isoindole-1,3(2h)-dione-4,7-ethano-
3a,4,7,7a-tetrahydro-2-
rt= 10.365
mw= 253.110279
pharmacological activity: antiinflammatory
table 2. zone of inhibition (mm) of test different bioactive compounds and standard antibiotics of
medicinal plants to macrophomia phaseolus.
s. no. plant zone of inhibition (mm)
1. ricinus communis (alkaloids) 2.99±0.19
2. datura stramonium(alkaloids) 6.61±0.26
3. linum usitatissimum (crude) 4.97±0.23
4. anastatica hierochuntica (crude) 5.98±0.22
5. cassia angustifolia (crude) 5.51±0.24
6. euphorbia lathyrus (crude) 6.00±0.25
7. rosmarinus oficinalis (crude) 5.54±0.23
8. citrullus colocynthis (crude) 4.10±0.17
9. althaea rosea (crude) 5.71±0.20
10. coriandrum sativum (crude) 3.42±0.21
11. origanum vulgare (crude) 5.63±0.25
12. urtica dioica (crude) 3.98±0.21
13. foeniculum vulgare (crude) 2.94±0.19
14. ocimum basilicum (crude) 5.03±0.23
15. control 0.00
cont... table 1. bioactive chemical compounds identified in methanolic extract of macrophomia phaseolus.
results and discussion
microscopical characteristics of fungal strains
were determined using specific media light and
compound microscope. gas chromatography and mass
spectroscopy analysis of compounds was carried out in
methanolic extract of macrophomia phaseolus, shown in
table 1. many compounds are identified in the present
study. some of them are biological compounds with
antimicrobial activities. clinical pathogens selected for
antibacterial activity namely, escherichia coli, bacillus
subtilis, pseudomonas eurogenosa, streptococcus
pyogenes, staphylococcus epidermidis, proteus
mirabilis, klebsiella pneumonia and staphylococcus
aureus, maximum zone formation against proteus
mirabilis 6.38±0.22 mm. methanolic extraction of
macrophomia phaseolus showed notable antifungal
activities against saccharomyces cerevisiae, m. canis,
384 indian journal of public health research & development, march 2018, vol. 9, no. 3
trichoderma viride, aspergillus flavus, aspergillus
fumigatus, penicillium expansum, aspergillus terreus
and candida albicans. aspergillus flavus was very highly
active against macrophomia phaseolus 6.009±0.23.
in agar well diffusion method the selected medicinal
plants were effective against macrophomia phaseolus
table 2. five-millimeter diameter wells were cut from
the agar and 20 ?l of the samples solutions ricinus
communis (alkaloids), datura stramonium(alkaloids),
linum usitatissimum (crude), 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). datura stramonium
(alkaloids) was very highly active 6.61±0.26 mm against
macrophomia phaseolus. macrophomia phaseolus was
found to be sensitive to all test medicinal plants and
mostly comparable to the standard reference antifungal
drug amphotericin b and fluconazole to some extent 39-
44. recently, it was demonstrated that volatile organic
compounds (vocs) of bacteria such as terpenoids,
phenylpropanoids and fatty acid 45-50 derivatives can
influence the growth of some fungi and, in general, the
inter- and intra-organismic communication signals.
conclusion
macrophomia phaseolus produce many important
secondary metabolites with high biological activities.
macrophomia phaseolus was found to be sensitive to
all test medicinal plants and mostly comparable to the
standard reference antifungal drug amphotericin b and
fluconazole to some extent.
financial disclosure: there is no financial
disclosure.
conflict of interest: none to declare.
ethical clearance
these experiments were carried out in accordance
with approved guidelines and all protocols were
approved under the department of biology, college of
science, hillah city, iraq.
references
1. mohammed gj,, kadhim mj, hameed ih. proteus
species: characterization and herbal antibacterial: a
review. international journal of pharmacognosy and
phytochemical research. 2016 8(11): 1844-1854.
2. gilani ah, janbaz kh, akhtar ms. selective
protective effect of an extract from fumaria
parviflora on paracetamol induced hepato-toxicity.
gen. pharmacol. 1996 27: 979-983
3. shireen sk,, hameed ih,, hamza lf. acorus
calamus: parts used, insecticidal, anti-fungal,
antitumour and anti-inflammatory activity: a
review. international journal of pharmaceutical
quality assurance. 2017 8(3): 153-157.
4. huda ja, hameed ih, hamza lf. anethum
graveolens: physicochemical properties, medicinal
uses, antimicrobial effects, antioxidant effect, antiinflammatory
and analgesic effects: a review.
international journal of pharmaceutical quality
assurance. 2017 8(3): 88-91.
5. hussein hm, hameed ih, ubaid jm. analysis of the
secondary metabolite products of ammi majus and
evaluation anti-insect activity. international journal
of pharmacognosy and phytochemical research.
2016 8(8): 1192-1189.
6. hussein hm, ubaid jm, hameed ih. inscticidal
activity of methanolic seeds extract of ricinus
communis on adult of callosobruchus maculatus
(coleopteran:brauchidae) and analysis of its
phytochemical composition. international journal of
pharmacognosy and phytochemical research. 2016
8(8): 1385-1397.
7. ubaid jm, hussein hm, hameed ih. determination
of bioactive chemical composition of callosobruchus
maculutus and investigation of its anti-fungal
activity. international journal of pharmcognosy and
phytochemical research. 2016 8(8): 1293-1299.
8. ibraheam ia, hussein hm, hameed ih. cyclamen
persicum: methanolic extract using gas
chromatography-mass spectrometry (gc-ms)
technique. international journal of pharmaceutical
quality assurance. 2017 8(4) 200-213.
9. ibraheam ia, hadi my, hameed ih. analysis of
bioactive compounds of methanolic leaves extract
of mentha pulegium using gas chromatography-
mass spectrometry (gc-ms) technique.
international journal of pharmaceutical quality
assurance. 2017 8(4) 174-182.
10. hadi my, hameed ih, ibraheam ia. ceratonia
siliqua: characterization, pharmaceutical products
indian journal of public health research & development, march 2018, vol. 9, no. 3 385
and analysis of bioactive compounds: a review.
research journal of pharmacy and technology.
2017 10(10): 3585-3589.
11. hadi my, hameed ih, ibraheam ia. mentha
pulegium: medicinal uses, anti-hepatic,
antibacterial, antioxidant effect and analysis of
bioactive natural compounds: a review. research
journal of pharmacy and technology. 2017 10(10):
3580-3584.
12. kadhim mj, sosa aa, hameed ih. evaluation of
anti-bacterial activity and bioactive chemical analysis
of ocimum basilicum using fourier transform
infrared (ft-ir) and gas chromatography-mass
spectrometry (gc-ms) techniques. international
journal of pharmacognosy and phytochemical
research. 2016 8(6): 127-146.
13. mohammed gj, kadhim mj, hussein hm.
characterization of bioactive chemical compounds
from aspergillus terreus and evaluation of
antibacterial and antifungal activity. international
journal of pharmacognosy and phytochemical
research. 2016 8(6): 889-905.
14. hameed ih, altameme hj, idan sa. artemisia
annua: biochemical products analysis of methanolic
aerial parts extract and anti-microbial capacity.
research journal of pharmaceutical, biological and
chemical sciences. 2016 7(2): 1843- 1868
15. jasim h, hussein ao, hameed ih, kareem
ma. characterization of alkaloid constitution
and evaluation of antimicrobial activity of
solanum nigrum using gas chromatography mass
spectrometry (gc-ms). journal of pharmacognosy
and phytotherapy. 2015 7(4): 56-72.
16. hadi my, mohammed gj, hameed ih. analysis
of bioactive chemical compounds of nigella sativa
using gas chromatography-mass spectrometry.
journal of pharmacognosy and phytotherapy. 2016
8(2): 8-24.
17. shareef hk, muhammed hj, hussein hm,
hameed ih. antibacterial effect of ginger (zingiber
officinale) roscoe and bioactive chemical analysis
using gas chromatography mass spectrum. oriental
journal of chemistry. 2016 32(2): 20-40.
18. al-jassaci mj, mohammed gj, hameed ih.
secondary metabolites analysis of saccharomyces
cerievisiae and evaluation of antibacterial activity.
international journal of pharmaceutical and clinical
research. 2016 8(5): 304-315.
19. mohammed gj, al-jassani mj, hameed ih. antibacterial,
antifungal activity and chemical analysis
of punica grantanum (pomegranate peel) using gcms
and ftir spectroscopy. international journal
of pharmacognosy and phytochemical research.
2016 8(3): 480-494.
20. dhahir bm, hameed ih, jaber ar. prospective
and retrospective study of fractures according
to trauma mechanism and type of bone fracture.
research journal of pharmacy and technology.
2017 10(10):1827-1835.
21. hapeep ma, hameed ih, jasim aa. risk factors,
cause and site of firearm injuries: a prospective
and retrospective study. research journal of
pharmacy and technology. 2017 10(10): 3420-
3425.
22. jasim aa, hameed ih, hapeep ma. traumatic
events in an urban and rural population of
children, adolescents and adults in babylon
governorate - iraq. research journal of pharmacy
and technology. 2017 10(10): 3429-3434.
23. altameme hj, hameed ih, abu-serag na.
analysis of bioactive phytochemical compounds
of two medicinal plants, equisetum arvense and
alchemila valgaris seed using gas chromatographymass
spectrometry and fourier-transform infrared
spectroscopy. malays. appl. biol. 2015 44(4):
47–58.
24. hussein hm, hameed ih, ibraheem oa.
antimicrobial activity and spectral chemical analysis
of methanolic leaves extract of adiantum capillus-
veneris using gc-ms and ft-ir spectroscopy.
international journal of pharmacognosy and
phytochemical research. 2016 8(3): 369-385.
25. kadhim mj, mohammed gj, hameed ih. in vitro
antibacterial, antifungal and phytochemical analysis
of methanolic fruit extract of cassia fistula. oriental
journal of chemistry. 2016 32(2): 10-30.
26. jaddoa hh, hameed ih, mohammed gj. analysis
of volatile metabolites released by staphylococcus
aureus using gas chromatography-mass
spectrometry and determination of its antifungal
activity. oriental journal of chemistry. 2016 32(4):
8-24.
27. hameed ih, salman hd, mohammed gj. evaluation
of antifungal and antibacterial activity and
386 indian journal of public health research & development, march 2018, vol. 9, no. 3
analysis of bioactive phytochemical compounds of
cinnamomum zeylanicum (cinnamon bark) using
gas chromatography-mass spectrometry. oriental
journal of chemistry. 2016 32(4): 16-25.
28. kadhim mj, mohammed gj, hussein hm. analysis
of bioactive metabolites from candida albicans
using (gc-ms) and evaluation of antibacterial
activity. international journal of pharmaceutical and
clinical research. 2016 8(7): 655-670.
29. ubaid jm, hussein hm, hameed ih. analysis of
bioactive compounds of tribolium castaneum and
evaluation of anti-bacterial activity. international
journal of pharmaceutical and clinical research.
2016 8(7): 655-670.
30. hameed, i.h., al-rubaye a.f. and kadhim, m.j.
antimicrobial activity of medicinal plants and
urinary tract infections. international journal of
pharmaceutical and clinical research. 2017 8(11):
44-54.
31. kadhim wa, kadhim, m.j., hameed, i.h.
antibacterial activity of several plant extracts
against proteus species. international journal of
pharmaceutical and clinical research. 2017 8(11):
88-94.
32. ahmed md, hameed ih, abd-ali mq. prospective
and retrospective study of the acute heart attack
cases in marjan hospital-hillah city-iraq. research
journal of pharmacy and technology. 2017 10(10):
3408-3416.
33. fakhir df, hameed ih, flayyih ss. burns injuries:
a prospective statistical study of 112 patients.
research journal of pharmacy and technology.
2017 10(10): 3401-3407.
34. mekhlef ak, hameed ih, khudhair me. prevalence
of physical injuries on the head, neck and entire
body in, hilla, iraq. research journal of pharmacy
and technology. 2017 10(10): 3276-3282.
35. kadhim mj. in vitro antifungal potential of
acinetobacter baumannii and determination of its
chemical composition by gas chromatography-mass
spectrometry. der pharma chemica. 2016 8(19):
657-665.
36. hameed ih, al-rubaye af, kadhim mj.
antimicrobial activity of medicinal plants and
urinary tract infections. international journal of
pharmaceutical and clinical research. 2017 9(1):
44-50.
37. al-rubaye af, hameed ih, kadhim mj. a review:
uses of gas chromatography-mass spectrometry
(gc-ms) technique for analysis of bioactive
natural compounds of some plants. international
journal of toxicological and pharmacological
research. 2017 9(1) 81-85.
38. kadhim mj, kaizal af, hameed ih. medicinal
plants used for treatment of rheumatoid arthritis:
a review. international journal of pharmaceutical
and clinical research. 2016 8(12): 1685-1694.
39. hussein hm, hameed ih, ubaid jm. analysis of the
secondary metabolite products of ammi majus and
evaluation anti-insect activity. international journal
of pharmacognosy and phytochemical research.
2016 8(8): 1192-1189.
40. ubaid jm, hussein hm, hameed ih. determination
of bioactive chemical composition of callosobruchus
maculutus and investigation of its anti-fungal
activity. international journal of pharmcognosy and
phytochemical research. 2016 8(8): 1293-1299.
41. hussein jh, hameed ih, hadi my. using gas
chromatography-mass spectrometry (gc-ms)
technique for analysis of bioactive compounds
of methanolic leaves extract of lepidium sativum.
research journal of pharmacy and technology.
2017 10 (11): 3981-3989.
42. hadi my, hameed ih. uses of gas chromatography-
mass spectrometry (gc-ms) technique for
analysis of bioactive chemical compounds of
lepidium sativum: a review. research journal of
pharmacy and technology. 2017 10 (11): 4039-
4042.
43. ubaid jm, hadi my, hameed ih. bioactive
chemical compounds identified in methanolic
extract of trogoderma granarium. research journal
of pharmacy and technology. 2017 10 (11): 3997-
4004.
44. hameed ih, calixto mr, hadi my. antimicrobial,
antioxidant, hemolytic, anti-anxiety, and
antihypertensive activity of passiflora species.
research journal of pharmacy and technology.
2017 10 (11): 4079-4084.
45. hameed ih, calixto mr, hadi my. a review:
solanum nigrum l. antimicrobial, antioxidant
properties, hepatoprotective effects and analysis
of bioactive natural compounds. research journal
indian journal of public health research & development, march 2018, vol. 9, no. 3 387
of pharmacy and technology. 2017 10 (11): 4063-
4068.
46. hussein jh, hameed ih, hadi my. a review:
anti-microbial, anti-inflammatory effect and
cardiovascular effects of garlic: allium sativum.
research journal of pharmacy and technology.
2017 10 (11): 4069-4078.
47. flayyih ss, hameed ih, fakhir fd. road traffic
accident coming to hillah teaching hospital:
prospective and retrospective study. research
journal of pharmacy and technology. 2017 10 (11):
3819-3825.
48. fakhir df, hameed ih, flayyih ss. retrospective
study: burn injury from 2010 to 2015 in a burn
unit-hillah teaching hospital-iraq. research
journal of pharmacy and technology. 2017 10 (11):
3831-3838.
49. khudhair me, hameed ih, mekhlef ak. a
prospective and retrospective study of acute
bronchitis in hillah city-iraq. research journal of
pharmacy and technology. 2017 10 (11): 3839-
3844.
50. kamal sa, hamza lf, ibraheam ia. characterization
of antifungal metabolites produced by aeromonas
hydropinghila and analysis of its chemical compounds
using gc-ms. research journal of pharmacy and
technology. 2017 10 (11): 3845-3851.