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

FATIMA ZOHRA HAMDANI1 & RACHIDA ALLEM2

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#,

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9 IASET

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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&(

NiK!isease severity(

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 "otatotuber 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

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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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http://faostat3.fao.org/faostat-gateway/go/to/home/F




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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

A!a/

Ci"#

Si(!(i

A!a/

Ci"#

A#a("i#

A!a/

5 he&en' 1'ol 3.. 7(1 ' ' *(-1

]'$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"inen'7'ol 11 12(-7 *(18 2(17 *(75

] $er"ineol 113- 15(57 *(1* *(* 1(**

Citronellol 122. 17(5. ' 1(*8 '

Citral 1287 17(* ' ' *(7.

1,.'octadien'5'ol,5,'

di#ethyl123* 17(3 *(11 8(-

2,.'octadienal,5,' 15*1 18(78 ' *(22 *(18

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di#ethyl

$hy#ol 15.* 1.(-0.30 -

'

_':le#ene 15* 1(25

- -

*(*8

Cis-2,6-Dimethyl-

2,6octadiene

1532 1(85- 0.45

'

Benzoic acid 2-

amino- methyl

15-2 1(-0.09 -

'

2,6-octadien-1-

ol,3,7dimethyl

15-7 1(37- 0.22

*(--

Copaene17*3 13(2*

- -'

!" β #lemene15-5 13(78

- 0.44'

#" β - #lemene15-3 13(1

- $.97*(*3

Ca%yophylene171 1-(71

- 3.4$2(.2

&ethyl '-

methylanth%anilate

1713 1-(734.02 -

'

(%omadend%ene175- 1-(-5

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#" β )amesene1783 2*(2*

0.65*(12

α -h*m*lene177 2*(2-

- 1.33*(5-

β -+elinene1738 21(15

- 0.47'

#lemene17-3 21(58

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α -)a%nesene18*- 21(7- ' *(.1 '

-cadinene

1815 21(3*

- 0.56

*(*1

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Eerolidol 1857 22(-8 ' ' *( 5

C aryo"hylene o&ide 1.*. 25(8**(*- *(25

*(*7

< Cadinol 1.87 28(2.' '

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= Sinensal1*. 2.(*7

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< Sinensal18* 2(2

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= arnesol218* 55(3-

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2

Phytol213 57(5.

' '*(*8

onoter"nes hydrocarbonXs )+ .5(8 5(.7 18(88

#onoter"nes o&ygXnXs )+ 57(-3 .,*8 1(38

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Fi#! 1: C=%a"%a %+ E!("ia) Oi) %+ Ci"# L!a;! A: Citrus reticulata B)a(-%

B: Citrus sinensis Osbeck; C: Citrus aurantium aaa

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6.2500000000000056#-2

0

20

40

60

$0

100

Citrus sinensis

Citrus reticulata

Citrus aurantium

Concentrations (mg/ml)

severity index of the disease(%)

01020304050

6070

Citrus sinensis

Citrus reticulata

Citrus aurantium



0204060$0

100120

Citrus sinensis Citrus reticulata Citrus aurantium



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C: T=! Sa! Ti!. Da"a A! M!a( S"a($a$ D!;ia"i%( E% Ba/

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28

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

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