Revista Mexicana de Micología ISSN: 0187-3180 [email protected]Sociedad Mexicana de Micología México Valenzuela-Cota, Daniel Fernando; Buitimea-Cantúa, Génesis Vidal; Rosas-Burgos, Ema Carina; Cinco-Moroyoqui, Francisco Javier; Yépiz-Gómez, María Susana; Cortez-Rocha, Mario Onofre; Plascencia-Jatomea, Maribel; Burgos-Hernández, Armando The antifungal effect of Jacquinia macrocarpa plant extracts on the growth of Aspergillus flavus, A. parasiticus and Fusarium verticillioides Revista Mexicana de Micología, vol. 39, junio, 2014, pp. 2-11 Sociedad Mexicana de Micología Xalapa, México Available in: http://www.redalyc.org/articulo.oa?id=88342643002 How to cite Complete issue More information about this article Journal's homepage in redalyc.org Scientific Information System Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Non-profit academic project, developed under the open access initiative
Efecto antifúngico de los extractos de la plantaJacquinia macrocarpa en eldesarrollo de Aspergillus flavus, A. parasiticus y Fusarium verlid//ioides
Resumen. El objetivo de este estudio fue evaluar el efecto de fracciones obtenidas de la planta}acquinia macrocarpa contra los hongos fitopalógenos Aspergillus flavus, A. parasiticus yFusarium verticillioides. Polvos de la planta deshidratada fueron extrafdos con metanol (70 %v/v)y partidonados con hexano, acetato de etilo yn-butano!. La fracción con mayor actividad contrael crecimiento radial de los hongos fue adicionalmente fraccionada por cromatografía en sflicagel. La fracción particionada en n-butanol (BF) mostró la mayor actividad antifdngica contra lostres hongos. La sub-fracción F5,4 inhibió el crecimiento radial de F. verticillioidescompletamente, mientras que el de A flavus y A parasiticus fue inhibido en 70 %Y 64 %,respectivamente. La germinación de esporas de A. parasiticus y F. verticillioides fue inhibida en7.B %Y11.6 %, respectivamente. La longitud yel diámetro de las hifasde los tres hongos fueronmenores que los de los controles. El diámetro de las esporas de las especies deAspergillus fueronreducidas, mientras que las esporas de F. verticillioides no fueron afectadas. La viabilidad de Aflavus, A. parasiticus y F. verticillioides fue reducida en 40.7 %, 44.6 % Y 46.3 %,respectivamente. La fracción F5.4 inhibió el desarrollo de los tres hongos significativamente,principalmente aF. verticillioides.Palabras clave: actividad antifúngica, patógenos del mafz, extractos de plantas
Abotract. The aim of this study was to evaluate lhe effect of fractiono obtained from the plant}acquinia macrocarpa against phytopathogenic Iongi Aspergillus flavus, A. parasiticus andFusarium verticillioides. The powder of the dried plant materials was extracted wilh melhanol(70 %v/v) and partitioned wilh hexane, elhyl acetate, and n-butanol. The most active fractionagainst lhe radial growth of lhe fungi was further fractionated by silica gel chromatography. Thefraetion from}. macrocarpa partitioned in n-butanol (BF) showed lhe highest antifungal aetivityagainst lhe lhree fungi. Sub-fraction F5,4 completely inhibited lhe growth of F. verticillioides,whereas lhe radial growth of A. flavus and A parasíticus was inhibited 70 % and 64 %,respectively. Spores germination of A parasiticus and F. verticillioides was inhibited 7.6 %and11.6 %, respectively. The Iengths and diamete.. of hyphae of lhe lhree fungi were smaller thanlhose of controlo. Spore diarnete.. of lhe Aspergillus species were reduced, while spores of F.verticillioides were not affeeted. The spore viability of A. flavus, A parasiticus and F.verticillioides was reduoed 40.7 %, 44.6 %and 46.3 %, respectively. Fraelion F5.4 significantlyinhibited the developmentoflhethree fungi, mainly F. verticillioides.Keywords: antifungal aelivity, maize palhogens, plantextracts
Recibido 7 de mayo 2013; aceptado 21 de marzo 2014.Received 7 May 2013; accepted 21 March 2014.
control PDA and R, is lhe colony radius value of eolonies
grown io PDA media containing plant extracts taking ioto
account lhe solvent effect (plasceneia-Jatomeaelal., 2(03).
(MIClO)' and 1he minimum lethal concen1ration (MLC) 1hat
inhibited 100 % of radial growth and tbat did not succeed
when it was transferred lo fresh culture media contaioing no
antifungal fraction (Rosas-Burgos el al., 2009). The
experimentwas ron io triplicate.
The radial growth iohibition percentages were
calculatedusiog lhe followiog formula:
Radial inhibition (%F X lOO
control containing melhanol.
Hyphal diameter and length
Petri dishes wilh potato dextrose agar (PDA) containing 3.0
mg mL-' of lhe antifungal fraction F5.4 was centrally poiot
iooculated wilh 1 x 10' spores mL" from 7-day-old cultures of
the fungi. Afier 72 h of incubation, one hundred
measurements ofdiameters and lenglhs ofapical hypha were
made on mycelia. The measurements were carried out usiog
lhe lmag.....Pro Plus version 6.3 software (Media Cybemetics,
Inc.) as reported by Larralde el al. (1997) and Cox el al.
(1998). One hundred measurements were also made on
mycelia grown io lhe control plates. The experiment was
repeated twice.
Where Se represents lhe percentage of germioated
spares in samples treated wilh lhe antifungal fraction F5.4,
and Se is lhe percentage ofgermioated spores observed io lhe
Klnet1cs ofspores germination
PDA plates containing 5 mg mL-' of solids of lhe antifungal
fraction F5.4 were iooculated by spreading 20 /lL ofa spare
suspension containing 1 x 10' spores onto lhe agar surface.
The plates were iocubated at 27 oC lo grow Aspergillus
species or 25 oC to grow R verticillioides. The number of
germioated spores per plate was determioed every 3 h by
countiog 200 spores (germioated and non-germioated) usiog
a light microscope. A spore was considered as germioated
when lhe lenglh of its germioal tube reached one-halfof lhe
spore diameter (paul el al., 1993, Plascencia-Jatomea el al.,
2(03). Each germioation assay was made io duplicate. l\vo
types of controls were prepared, one contaioed PDA media
plus melhanol, and lhe olher one contaioed onIy PDAmedia.
The iohibition ofspore germioation was calculated using lhe
followiog equation:
Spores diameter
Spores diameter was determined accordiog to lhe procedure
of Rarris (1999). Coverslips were placed io Petri plates and
covered with 10 mL of potato dextrose broth (PDB)
containing 5.0 mg mL-' ofF5.4. Two types of controls were
prepare<!, one contaioed PDB plus a volume of methanol
equal lo lhat used to dissolve lhe solids of lhe antifungal
fraction, and lhe olher one contaioed only PDB. The plates
were iooculated wilh20 /lLofa I x lO' sporesuspensionofA.
flavus, A. parasilicus. or R verlicillioides, and their
development was monitored until they germioated. One
coverslip containing spores was removed at random every 3 h
from the plates and 100 measurements ofspore diameter were
carried out usiog the lmag.....Pro Plus version 6.3 (Media
Cybernetics, Inc.). For F. verlicillioides .pores, the
measurements were made wide- and length-wise. The
experiment was repeated twice.
Spores viability
Spores viability analysis ofthe fungi was made according the
methodology described by Granjo el al. (2007). Discs of
sterilized cellophane measuring 2.5 cm io diameter were
X lOOGermioation inhibition (%) ~
distributed wilh sterile forceps on lhe surface of a Petri disb
containing solidified PDA medium plus S mg mL-' of FS.4.
Two types ofcontrols wilhno cellophane discs were prepared,
one contained PDAmedia plus a volume ofmelhanol equal lo
!hat used lo dissolve lhe solida of lhe antifungal fraction,
whereas lhe olher one contained only PDA media. The
cellophane discs were inoculated wilh SO ¡LLofa IxlO'spore
suspension of each fungus and incubated at 27 oC lo grow
Aspergillus species or at 2S oC lo grow R vertieillioides. The
cellophane discs were incubated by 0,12,18,24,48,72 Y96
h. At lhe end of each incubation time, each cellophane disc
was removed from lhe Petri dish and placed over a
microscope slide and covered wilh equal volumes of fiual
working solutions of FDA [3,6' diacetyl fluorescein-FDA
(Sigma Chemical Co., SI. Louis, MO, USA); S mg mL-'
acetone and diluted 2S00 times in phosphate-bufered saline
(PBS), pH 7.4 at lhe time ofuse] and EB [2.7-diamino-lO
elhyl-9-phenyl phenanthridine (Sigma Chemical Co.); I g
mL-' in PBS and diluted 20 times in PBS at lhe time of use]
(Granjo et al., 2007). The lreated slides were incubated at 2S
oC for 30 min, covered wilh a coverslip, and observed under a
fluorescence microscope (Leica DM 2S00). One hundred
spores were analyzed for each incubation time and lhe
proportion of lhe green-colored viable spores and lhe red
colored non-viable spores was determined. The experiment
was repeatedtwice.
Statisticalanalysis
Analysis ofdata was carried out by ANOVA using a factorial
desigu (plant and extraction solvent). Comparison ofmeans
was performed by lhe Tukey test (P<O.OS) using lhe SAS
program (SAS, 200S).
~ResultsRadial eItension growth
The crude melhanolic extraet from J. macrocarpa (CE) and
every partitioned fraction showed radial growth inhibition
against lhe three fungi (P<O.OS), wilh major inhibition effect
against R vertieillioides (Table 1). The n-butanol fraction
(BF) showed lhe highest radial growth inhibition against lhe
!bree fungi. R vertieillioides did not show statistical
differences (P<O.OS) in inhibition growth by BF and CE.
Similarly, no statistical differences (P<O.OS) were also
observed in inhibition growth ofA. parasiticus by AEF and
WF, whereas lhe radial growth inhibition values ofA. jlavus
by CE and all fractions differed siguificantly (P<O.OS). The
antifungal fractions obtained in lhe different stapes of
purification (FS and FS.4) showed high radial growth
inhibition activity against lhe three fungi in comparison lo
lhose oflhe partitioned fractions and CE. The radial growthof
R ver/ieillioides was lotally inhibited when it was inoculated
onPDAcontaining fractions FS andFS.4.
MGI,MIC"andMLCofBF
The three fungi sbowed gradual radial growth inhibition when
lhe BF concentration in lhe PDAmedia was increased (Table
2). The MGI for A.jlavus andA. parasiticus was S.O mg mL-',
which caused lhat lhe fungi did not reach SO % of radial
growth inhibition. R ver/ieillioides showed MGI and MIC",
values of 4.0 mg mL-' and 1.0 mg mL-', respectively. In lhe
presence ofBF, lhe radial growth inhibition ofA.jlavus andA.
parasitieus were 70 % and 64 %, respectively. However,
when R vertieillioides was inoculated on PDA media
containing lhe fractions obtained from BF, ita radial growth
was completely inhibited (Table 1) allhough was able lo grow
when it was transferred lo PDB containing no antifungal
fraction. The influence of lhe variable concentration of lhe
....l<1Z~
¡;;:O
•
Table l. Inhibition ofradial growth (%) of fungi by severa! Jacquinia macrocarpa fractions
Trea1ment' A.j1avus A. parasiticus F. verticillio ides
CE 36.0±O.06' 19.0±0.06' 94.4±0.07·
HF 34.4±O.05' 14.0±0.07' 66.8±o.05'
BF 40.0±O.IO· 38.0±032· 94.4±0.10·
AEF 232±O.l3' 24.8±0.06b 58.8±0.l3'
WF 332±1.06" 252±0.05b 72.0±1.06b
F5 62.0±O.0 48.0±0.0 100.O±O. O
F5.4 70.0±O.0 64.0±0.0 100.O±O. O
'CE~crude extrael, HF~Hexan fraction, BF~-botanol fraction, AEF~thyl acelate fractioo, WF~ter fraetioo, FS~tion obtained bycolumn ebromatography in siliea gel from BF, FS.<!='fractioo obtainedby!hin layer ebromatography from FS. Volues are !he average ofthreereplicates ± standard error. Al1 values were corrected considering tbe solvent effect. Values with different letter in the same column aredifferent (P<O.OS).
Table 2. Inhibition ofradial growth (%) offungi after 336 h ofincubation in different concentrations ofthe n-butanol fraction ofJacquinia macrocarpa
Solids (mg'mL) A.j1avus A. parasiticus F. verticillioides
1.0 22.7±0.O' 0.0 ±O.O' 63.6±0.O'
2.0 31.8±0.0' l3.6±O.O' 77.3±0.0'
3.0 36.4±0.Ob 22.7±o.O' 81.5±0.Ob
4.0 36.4±0.Ob 31.8±0.Ob 86.4±0.0·
5.0 40.9±0.7· 36.4±0.0' 86.4±0.0·
Volues are tbe average of three replieates ± standsrd error. Volues with different letter in tbe same eolumn are different (P<O.OS).
antifungal fraction BF over the three fungi is shown in Figure respeclively).
1.
Hyphal diametcr and length
Kinetics ofspores germination The three fungi sigoificantly (P<O.OS) uoderwent a reduction
The spores ofthe three fungi inoculatOO on the PDB controls in diameter and length of apical hyphae when they were
eompletely germinated within 30 h. Although spores grown on PDA agar media containing the F5.4 fraction in
germination ofA. j1avus was delayed when they were grown respeet lo the PDA and methanol controls. A. j1avus apical
on PDA containing the antifungal F5.4 fractiort, all spores hyphaeshowedareduetionindiameterandlengthin20.8±1.8
wereabletogerminateattheendoftheexperiment(Figure2). % and 43.8±2.1 %, whereas those for A. parasiticus were
SimilarresultswereobservedforA.j1avus,A.parasiticusand reduced in 18.S±O.8 % and 40.1±1.7 %, respectively. The
F. verticillioides spores, which were delayed in growth on decrease in diameter and length of F. verticillioides apical
PDA containing the F5.4 fraetion (Figure 2). However, the hyphae was of 18.2±1.6 % and41.4±2.6 %, respectively. The
spores of the two last fungi showed sigoificant (P<0.05) three fungi showed the higher (P<0.05) reduclion percentage
germinationinhibitionatthe endofthe incubationtime (7.8 % inlength than indiameter.
and 11.8 % for A. parasiticus and F. verticillioides,
......DiScussion
Aspergillus flavus
Aspergillus parasiticus
Fusarium verticillioides
Figure 1. Radial growth ofAspergillus flavus, A. parasiticus and Fusarium verticillioides over potato dextrose agar media (PDA)containing n-butanol partitioned fraction ofJacquinia macrocarpa (BF) at concentrations of: A) OmglrnL, B) 1 mglrnL, C) 2 mglrnL,D) 3 mglrnL, E) 4 mglrnL, andF) 5 mglrnL.
Sporesdiameter andF. verticillioides was reduced in 40.7±1.8 %, 44.8±1.9 %
Diameter ofspores ofeach fungi were measured every 3 h of and 46.3±0.9 %, respectively.
incubation. In both Aspergillus species, the spores diameter
increased when they were incubated in PDB and PDB
containing methanol, however the diameter of spores of
Aspergillus species were reduced in PDB containing the
antifungal fractionFS.4 (Figure 3). Spores ofF. verticillioides Fungal growth inhibition of few Jacquinia species has been
showed no significant differences (P<O.OS) in length or reported. Sharma et al. (2008) studied an ethanolic extract of
fungi such as A. flavus and F. moniliforme (syn. F.
Spores viability verticillioides). García-Sosa et al. (2011) reported antifungal
Spores ofthree fungi treated with the antifungal fraction FS.4 activity of a methanolic extract of J. flammea against
showed a significant viability reduction (P<O.OS) after 96 h of phytopathogenic fungi different to those evaluated in the
incubation. The spores viability of A. jlavus, A. parasiticus present study. Although, extracts from otherplants have been
....1<l:ZI.!:le:::O
tested in their antifungal activity against A. flavus, A.
250 parasiticus and R verticillioides (Dabur et al., 2005; Rosas-A. flavus
Burgos et al., 2009; Sánchez et al., 2005; Satish et al., 2007;200
00
Suárez-Jiménez et al., 2007; Tequida-Meneses et al., 2002;~o Vargas-Arispuro et al., 2005), no studies on the antifungal~ 150
"O1!
activity of J. macrocarpa have not been reported before.".~ 100¡¡
Results of the radial growth inhibition test for A. flavus were(J
50 similar to those reported by Tequida-Meneses et al. (2002),
although they differed to those reported by other authors10 20 30 40 50
(Cowen and Steinbach, 2008; Rosas-Burgos et al., 2009;
Sánchez et al., 2005; Satish et al., 2007; Vargas-Arispuro et
al., 2005).250
A. parasiticlIS Radial growth inhibition of A. parasiticus and R
200 verticillioides by methanolic plant extracts have also been00
~ observed by Sánchez et al. (2005) and Suárez-Jiménez et al.o~ 150
"O
(2007), respectively. The radial growth inhibition value ofR"o;""§ 100
verticillioides was the highest observed in aH fungi, which is"(J
50 in agreement with other authors (Tequida-Meneses et al.,
2002; Rosas-Burgos et al., 2009). Sharma et al. (2008)o
o 10 20 30 40 50 reported a similar inhibition effect against R moniliforme and
A. flavus of ethanolic petals extracts of J. ruscifolia. In the
present study, the fractions obtained after the250
chromatographic purification steps (FS and FS.4) from BF,'V F. verlicillioides.... showed the higher antifungal activity over the three fungi thanO 200N, 00 that of the crude extracts and partitioned fractions. It wasel' ~
oen ;r 150
<1 "O observed that R verticillioides was the most inhibited mold,- *t.::I t:
when it was inoculated on PDA containing the fraction FS orO "§ 100...JO "u
(J
FS.4. The concentration of BF that caused the maximumil 50
Lj.l radial growth inhibition of both Aspergillus species wase<1 o significantly higher (P<O.OS) than that for R verticillioides,z o 10 20 30 40 50<1
which confirms that this last fungus is very sensible to theu lncuhation time (h)><~ antifungal fraction ofJ. macrocarpa. The results ofthis study
¡::! indicate that J. macrocarpa contains antifungal compounds\;\
:> Figure 2. Spore germination kinetics ofA. flavus, A. parasiticus and that are active against the growth of the fungi tested in theLj.lel:: F. verticillioides, grown on PDA (e), PDA plus methanol (O), and
Retarded spore germination observed in the present
study was also reported in R verticillioides by methanolic
'.
16
14 A. parasiticus
~'":" 12
~..~ 10
'ti<JI
~ 6oc.Ul
....1<l:ZI.!:le:::O
10 20 30 40
14
F. verticillioidesF. verticü1ioides
13
Figure 3. Spore size ofA.jlavus,A. parasiticus andF. vertici/lioides, grown on potato dextrose agar (PDA) (-), PDAplus methanol (O), and
PDAplusF5.4 (T).
3530252015
Incubation time (h)
10
E:=.. 12
.J:.'el¡¡¡ 11
<JI
~:s. 10
Ul
30252015
Incubation time (h)
10
...CllQíE'"'ti<JICllo~ 2
E 4~
extraets ofBaccharis glutinosa (Suárez-Jiménez et al., 2007). B. glutinosa was previously observed by Rosas-Burgos et al.
More reeent1y, Rosas-Burgos et al. (2011) reported retarded (2011), although these authors observed a more pronouneed
germination of spores of A. jlavus, A. parasiticus and F. antifimgal effeet of the p1ant extraet. In the present study,
verticillioides by a purified fraetion of B. glutinosa. Rosas- spores of Aspergillus speeies ineubated in PDA eontro1s
Burgos et al. (2011) observed 10wer spore germination inereased in size, whi1e the spores ineubated in the presenee of
inhibition inA. parasiticus than those observed in the present the antifimgal F5.4 fraetion showed a reduetion. Rosas
study. However, those authors reported higher inhibition ofF. Burgos et al. (2011) reported that in A. jlavus and A.
verticillioides spores, whieh was not observed in this work. parasiticus the spores diameter inereased when they were
The high spore viability inhibition of A. parasiticus and F. ineubated in media eontro1s and media eontaining the B.
verticillioides (P<0.05) eoineided with high spore glutinosa antifimgal fraetion, although the inerease observed
germination inhibition, whereas the opposite was observed in was smaller with the antifimgal treatrnent in eomparison of
A. jlavus, that is, at 10wer number ofviab1e spores, the 10wer that ofthe eontro1s.
sporesnumberwithgerminationeapaeity. A study reported by Ha et al. (2006) indieated that
Reduetion in diameter and 1ength of apiea1 hyphae Fusarium solani f. sp. pisi, resistant to easpofimgin aeetate,
observed in the three fimgi treated with a purified fraetion of whieh target is the fimga1 wall, deereased its resistanee when
•
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The authors are grateful lo the Nationa1 Science Council and
Technology (CONACyT» for granting fimding forthis study
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