4. Applied - IJANS - Antifungal Activity of the Leaf Essential - Fatima Zohra Hamdani - Algeria -...

8/20/2019 4. Applied - IJANS - Antifungal Activity of the Leaf Essential - Fatima Zohra Hamdani - Algeria - OPaid
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ANTIFUNGAL ACTIVITY OF THE LEAF ESSENTIAL OIL OF CITRUS AGAINST
ALTERNARIA ALTERNATA IN VIVO
1Hassiba, Benbouali University, Institute of Agricultural Sciences, Production and
Plant Protection Laboratory in the Chlef region, Chlef, Algeria
2Hassiba, Benbouali University, aculty of Science, !e"art#ent of Biology
Laboratory of Bioresources local, Chlef, Algeria
ABSTRACT
$he essential oils %ere e&tracted by hydro'distillation fro# the leaves of citrus by using hydrodistillation( $he
largest yields %ere recorded %ith Citrus limon L )1(*2+ and Citrus sinensis )*(-.+(/C0S of the essential oil of Citrus
reveled li#onene )(13 45.(1*+, 6' "inene )7(58 ' 5*(*+ and linalool )*(21 4.5(*5+ as the "rinci"al #a9or co#"ounds(
:ssential oils of citrus e&hibit a strong inhibiting effect on the develo"#ent of Alternaria alternata on leaves and "otato(
Lo% concentrations generate the lo%est severity inde&es of disease for all citrus s"ecies( According to the results, the
essential oils of citrus could be used as "otential antifungal agents for the control of Alternaria alternata in vivo(
KEYWORDS: Citrus, :ssential ;ils, Alternaria Alternata, Potato , In Vivo Antifungal Activity
INTRODUCTION
$he "hyto"athogenic fungi are <no%n to cause several "lant diseases and yield losses for #any econo#ically
i#"ortant cro"s )letcher et al(, 2**.= Abdel>ader et al, 2*12(( In agriculture, each year there are about loss of efficiency
of about 2*+ #ainly due to fungal diseases )Ba9%a et al(, 2**7(
$he genus Alternaria includes sa"ro"hytes and "lant "athogens )$ho##a, 2**5( It has over 1** s"ecies
ubi?uitous e&tre#ely co##on in soils, vegetation, air or food )Cal#es, 2*11( !ue to their gro%th, even at lo%
te#"erature, Alternaria s"" are "athogens "ostharvest fa#iliar, res"onsible for food s"oilage during refrigerated trans"ort
and storage );stry, 2**3( $he econo#ic losses are #ainly related to the reduction in ?uality due to a decrease in the
nutritional value )>osia< et al(, 2**7(
Algeria is the second largest "otato "roducer in the Arab %orld and in Africa after :gy"t %ith a "roduction of
7(21-(7.(** ton in 2*12 )A;S$A$ 2*17( Alternaria alternata is one of "revalent "athogens causing bro%n leaf s"ot on
"otato %orld%ide )$ho##a, 2**5(
$he disease bro%n s"ots on leaves is <no%n as one of the #ost destructive and co##unes of "otatoes cultivated
in regions %ith fre?uent "reci"itation and high relative hu#idity conditions( $he disease can occur on a %ide range of
cli#atic conditions )Pitt and Hoc<ing, 2**-(
Studies %ere conducted in vitro and in vivo to evaluate the antifungal activity of essential oils of citrus and their
active co#"ounds( $hey re"orted their inhibitors "o%ers against so#e "hyto"athogenic fungi such as Alternaria, usariu#,
www.ia!".# !$i"%'ia!".#
ISSN0/: 21345146 ISSNE/: 2134522
V%). 47 I#! 47 *#( 3 *#) 25187 18324
9 IASET
A("i+#(a) A-"i;i"< %+ "=! L!a+ E!("ia) Oi) %+ Ci"# Aai(" A)"!(aia A)"!(a"a i( Vi;%
1>
Penicilliu# and As"ergillus )Shar#a and $ri"athi 2**3= @iuda'artos et al (, 2**3= Cosic et al , 2*1*=( /u#us et al( 2*1*=
Phili"s et al, 2*12= Ha#dani et al, 2*18= Ha#dani et alle#, 2*18( $his study ai#s to evaluate the antifungal activity of
essential oils of citrus in vivo against Alternaria alternata.
MATERIALS AND METHODS
0)a(" Ma"!ia)
resh leaves of Citrus sinensis, Citrus aurantium and Citrus reticulate %ere collected fro# the adult trees of the
orchards of Chlef, Algeria during ars )2*11(
F#(a) S"ai( U!$
Alternaria alternate %as "roviding fro# the Culture Collection of the laboratory of the "athology of the "lants,
Institute of agrono#ic sciences, university of Chlef( $he fungal strain cultures %ere #aintained on a Potato !e&trose Agar
)P!A at 22 2 C(
E?"a-"i%( %+ E!("ia) Oi)
resh leaves of Citrus sinensis, Citrus aurantium and Citrus reticulate %ere hydrodistilled in a Clevenger'ty"e
a""aratus for 5h to obtain essential oil( 5**g of leaves %ere in boiling %ater during distillation( $he obtained essential oils
%ere dried over anhydrous sodiu# sul"hate and <e"t at 7 C for other e&"eri#ents( ield of leaf essential oil %as 1(*2 +(
A(a)<@! GC MS
$he co#"osition of the oil %as analyDed using He%lett4Pac<ard .3-* gas chro#atogra"h and Agilent
$echnologies HP 8-5E #ass s"ectro#etry e?ui""ed %ith a HP8S ca"illary colu#n )5* # & *,28 ##, thic<ness of fil#
*,28 F #( ;ven te#"erature %as initially held .* C during 2 #in, then increased 2.* C at a rate of 8 C0#in and held
for 1* #in( Carrier gas %as heliu# at a Go% rate of 1 )#l 0#in( Co#"onents %ere identied by co#"aring their #ass
s"ectra %ith iley and EIS$ library data and standards of the #ain co#"onents( Juantication of co#"onents %as
obtained by integrating the "ea< area of the chro#atogra# )Ha#dani et al, 2*18 (
A("i+#(a) A-"i;i"< %( 0%"a"% L!a+
$he evaluation of the antifungal "otential of the essential oil in vivo %as deter#ined by direct contact, the
essential oil is added directly to the culture #ediu# to allo% good adhesion of the oil %ith all of the leaf area and reduce
the ris< of eva"oration of the essential oil )eng et al(, 2*11(
After disinfecting a surface follo%ed by several rinses %ith distilled %ater, leaflets %ere "laced in Petri dishes -
c# in dia#eter on %ater agar 7+ to ensure saturating hu#idity throughout the test(( Inoculation of the leaflets %as #ade
using 2* Fl of a s"ore sus"ension )1*8s"ores0#l divided into si& "oints( $he dilution of essential oil %as #ade in ethanol
)1* Fl0#l ethanol to "re"are solutions at different concentrations )1, *(8, *(28, *(128 and *(*.28 #g0#l(
$he essential oil %as tested using three treat#ents a""lication na#ely "reventive, curative and at the sa#e ti#e(
or "reventive tests, the essential oil is used 27 hours before inoculation of the leaflets by the s"ore sus"ension and it is
used 27 h after inoculation for curative tests( for the test at the sa#e ti#e, the essential oil is su""lied 8 #in after
inoculation %ith the s"ore sus"ension( Controls %ithout essential oils for each test %ere "re"ared in the sa#e as the
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1
treat#ent( (
$he Petri dishes are incubated in the dar< for 73 hours and after "laced in the light under a#bient laboratory
conditions ):l abdellaoui et al (, 2**8( :ach test is re"eated three ti#es( Scoring the disease severity is calculated by
reference to leaf area and esti#ated fro# the rating scale Eotteghe# et al( )1-3*( $he severity inde& is calculated using
the for#ulaK
IS )MNini0-EtO1**
ISK!isease severity inde&(
niKnu#ber of leafs %ith the severity of the disease(
EtK $otal nu#ber of leafs
A("i+#(a) A-"i;i"< %( W%#($3I(%-#)a"!$ 0%"a"% T#!
:&"eri#ents %ere conducted %ith co##ercial "otato tuber )Solanum tuberosum var S"unta of Chlef, Algeria(
resh "otato tubers %ere harvested at the #ature stage and sorted based on siDe and absence of "hysical in9uries or disease
infection( Surface "otato tubers %ere steriliDed and "re"ared for inoculation by inGicting a dee" rind %ound as described
by ):ntsar and Bada%y, 2*12( $he tubers %ere rando#ly distributed into grou"s of ten tubers and three re"licates %ere
used for each treat#ent( $he first treat#ents )sa#e ti#e tubers %ere di""ed in the essential oil solution of concentration 1
#g 0 #l diluted in ethanol for five #inutes follo%ed by a di""ing in the inoculu#s for 5 #inutes(
Preventative treat#ents %ere done by an initial a""lying of Citrus oil by s"raying )1*** 0#l to in9ured "otato tuber for five #inutes, 27 hours before soa<ing in the inoculu# for 5 #inutes )Plooy et al(, 2**-( Control treat#ents
consisted of s"raying the %ounded "otato tuber %ith sterile %ater only, before or after inoculation as relevant( Potato tubers
inoculated %ere incubated in an incubator at 2* 2 C( After fifteen days of incubation, they %ere revealed to be stored
under a#bient laboratory conditions for t%o #onths( After storage, the nu#ber of %ounds that sho%ed disease sy#"to#s
%as recorded and disease incidence )+ %as calculated as follo%sK
!isease incidence DI ( )=( number of infected wounds
total wounds per replicate ) x100
S"a"i"i-a) A(a)<i
ean values and standard deviations %ere calculated for all tests and %ere sub9ected to statistical analysis using
Statistica(*., Statistical "ac<age )Statsoft, 1--8( !ifferences bet%een #eans %ere tested using test LS! of isher
A(a)<i GC MS %+ "=! E!("ia) Oi) %+ Ci"#
:ssential oils are e&tracted fro# the sheets of Citrus sinensis, Citrus aurantium and Citrus reticulate by hydro
distillation( $he yields obtained are *(-., *( 81 and *(5+ res"ectively for Citrus sinensis, Citrus aurantium and Citrus
reticulata( $he ?ualitative and ?uantitative analysis of the che#ical "rofiles of essential oils is indicated in $able 1( $he
table in cludes the ti#e of retention and "ercentage of the 81co#"onents identified, accounting for -2(7 to -3(32+ of all
www.ia!".# !$i"%'ia!".#
1 Fa"ia Z%=a Ha$a(i & Ra-=i$a A))!
the co#"onents( Using a ca"illary colu#n HP8S, chro#atogra#s of three essential oils of citrus tested %ere obtained
and re"resented in figure 1($he out"uts obtained fro# the essential oils areK $he results obtained "roved that there are
?ualitative si#ilarities bet%een the four essential oils, although the ?uantities of so#e corres"onding co#"ounds are
different( 1* co#"onds %ere recorded 9ointly a#ong the 81 identified, %here li#onene )(13 45.(1*+, 6' "inene )7(58 ' 5*(*+ and linalool )*(21 4.5(*5+ re"resented the "rinci"al #a9or co#"ounds( $he greatest volatile fraction returns of
#onoter"enes hydrocarbons for Citrus sinensis, Citrus reticulata are 5(.7 and, .5(8 res"ectively( $he o&ygenated
#onoter"enes e&"lained a""ro&i#ately 1(38+ of the identified co#"onents of the essential oil of Citrus aurantium ($he
high "ercentage of #onoter"enes in essential oils of citrus has been re"orted by several studies )Baalioua#er 1-3=
Settanni et al, 2*12((/C0S of the essential oil of Citrus reticulata revealed the "resence of strong concentrations in
$er"inene )2.(.2+, li#onene )22(82+ and 6'"inene )7(58+ re"resenting 85(7-+ of the total surface of the "ea<)$able
1( $he essential oil of Citrus sinensis is characteriDed byhigh concentrations in 6'"inene )5*+, li#onene)-(5+ and the
"resence of t%o is o#ersses?uiter"enes )Q and ): 6 ' :le#ene %ith a"re"onderance of the iso#er ): 6 4 :le#ene
)3(-+( or the essential oil of Citrus aurantium, linalool ).5(*5+
A("i+#(a) A-"i;i"< %( 0%"a"% L!a+
$he virulence of Alternaria alternata %as confir#ed by the "resence of ty"ical s"ots on the leafs( $he results of
the effect of different concentrations of essential oils of Citrus sinensis, Citrus aurantium and Citrus reticulata are shown
in Figure 1(It sho%s that the essential oil of Citrus reticulata e&"oses a strong antifungal "o%er( $he sa#e inhibitory
"otency %as observed in vitro )Ha#dani et alle#, 2*18$he concentrations of *(*.28, *(128 and *(28 #g 0 #l generate
lo%est severity inde& of the disease(
$he concentrations of *(8 and 1 #g 0 #l involve the a""earance of the signs of "hytoto&icity and leafs have
translucid leaf surfaces of yello%'bro%n to dar< bro%n( $his condition increases the #ycelial gro%th of Alternaria
alternata(
eng and Qheng )2** note that the develo"#ent of Alternaria alternata %as inhibited by high concentrations of
essential oil of Cinnamomum aromaticumin vivoon to#ato( $ian et al( )2*11 obtain a reduction in the develo"#ent of
Alternaria alternata 35(5+ on cherry to#atoes treated %ith the essential oil of Anethum graveolens L(
$he necrotic s"ot %asreducing in "reventive treat#ent( Reducing of necrotic s"ots in "reventive treat#ent #ay be
attributed to the contact ti#e of the essential oil )27 h "rior to infection %ith the leaf %hich "ro#otes the increased
resistance of "lant against Alternaria alternata
Several studies #ention the contribution of essential oils in increasing "lant resistance against e&ternal aggression
and "rovide an i#"ortant defense strategy for "lants, in "articular against "arasitic insects herbivores and "hyto"athogenic
fungi)Langenhei#, 1--7= !ayan et al(, 2**-( $hey act as #olecular signals and illustrate the evolutionary lin<s %ith their
functional roles $heis :$ Lerdau, 2**5(
A("i+#(a) A-"i;i"< %( W%#($3I(%-#)a"!$ 0%"a"% T#!
$he results )igure 5 illustrate the inhibitory effect of essential oils a""lied in "reventive treat#ent co#"ared
%ith the control( It records disease inde&es in the order of *+ for essential oils of Citrus sinensis, Citrus aurantium and
Citrus reticulata., this inhibitory effect disa""ears %hen the essential oils are used in "rocessing si#ultaneously( Changes
in the sha"e and fir#ness acco#"anied by the a""earance of #ycelial gro%th at areas of in9ury %ere observed( Plooy
I,a-" Fa-"% *CC/: 2.48 NAAS Ra"i(: 2.4
1
et al (, )2**- by testing both "reventive and curative treat#ent, they notice that the a""lication of the essential oil Lippia
scaberrima "reventively reduced significantly the "ercentage of inoculated citrus fruit %ith Penicillium digitatum
co#"ared
CONCLUSIONS
:ssential oils of citrus e&hibit a strong inhibiting effect on the develo"#ent of Alternaria alternata on leaves and
"otato( Lo% concentrations generate the lo%est severity inde&es of disease for all citrus s"ecies( $he results obtained
suggest the use of essential oils in food "reservation( :ffectively, considerable interest has develo"ed on the conservation
of food by the e#"loy of essential oils to "ronounce the effect on slo%ing gro%th and li#iting the "roduction of
#ycoto&ins )S#ith et al(, 2**8= ;#idbeygi et al(, 2**
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A00ENDICES
Ta)! 1: C=!i-a) C%,%i"i%( %+ E!("ia) Oi) %+ Citrus sinensis, Citrus aurantium a($ Citrus reticulata
C%,%a(" KI RTMi(/
Ci"#
R!"i-#)a"a
]'$hu9ene -51 8(31 2(** 1(7- *(*2
< 'Pinene -5- 8(-- 7(5. 2(*7 *(53
camphene -85 .(5 *(1* ' *(*5
= ' Pinene -- (12 7(58 5*(* 8(28
= ' yrcene --1 (7. 1(1- 7(82 1(7*
< Phellandrene 1**3 (3. *(1* *(.3 '
5 Carene 1*12 3(*2 ' .(71 *(1.
)^'7'Carene 1*58 3(2* *(53 2( '
!'Li#onene 1*5. 3(1 22(82 -(5 (13
= ;ci#ene 1*88 3(7 ' *(7 '
1,5,.'octatriene,5,'
di#ethyl' 1** -(11 *(-7 3(8- 1(15
$er"inene 1*3* -(71 2.(.2 7(72 ' Cis sabinene hydrate 1*-7 -( ' *(21 '
$er"inolene 1*-. 1*(28 1(1. 2(85 '
Linalool 1123 1*(1 *(21 1(27 .5(*5
Q Allooci#ene 1182 11(53 *(*5 *(*3 '
Citronellal 118- 12(*. ' *(-* '
] $er"ineol 113- 15(57 *(1* *(* 1(**
Citronellol 122. 17(5. ' 1(*8 '
Citral 1287 17(* ' ' *(7.
www.ia!".# !$i"%'ia!".#
di#ethyl
'
- -
- $.97 *(*3
0.65 *(12
- 1.33 *(5-
-cadinene
Eerolidol 1857 22(-8 ' ' *( 5
*(*7
' *(5* '
I,a-" Fa-"% *CC/: 2.48 NAAS Ra"i(: 2.4
2
Ses?uiter"ne hydrocarbonX )+ ' 18(-8 5(..
Ses?uiter"nes o&ygXnXs )+ *(*- 1(28 *(78
autres )+ ' ' *(-.
$otal identifiX )+ -3(32 -.(3- -2(7
Fi#! 1: C=%a"%a %+ E!("ia) Oi) %+ Ci"# L!a;! A: Citrus reticulata B)a(-%
B: Citrus sinensis Osbeck; C: Citrus aurantium aaa
www.ia!".# !$i"%'ia!".#
6.2500000000000056#-2
0 10 20 30 40 50
60 70
Citrus sinensis
Citrus reticulata
Citrus aurantium
Concentrations (mg/ml)
100 120
Concentrations (mg/ml)
Fi#! 2: E++!-" %+ C%(-!("a"i%( %+ "=! E!("ia) Oi) %+ Ci"# %( "=! I($!? %+
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