Extraction of Bioactive Compounds from Luminescent Mushroom...

726Extraction of Bioactive Compounds from Luminescent Mushroom (Neonothopanus nambi)

and its Effect on Root-Knot Nematode (Meloidogyne incognita)วารสารวิจัย มข. 15 (8) : สิงหาคม 2553

Extraction of Bioactive Compounds from Luminescent Mushroom (Neonothopanus nambi) and its Effect on Root-Knot Nematode

(Meloidogyne incognita)

Sureeporn Bua-art1, Weerasak Saksirirat2*, Somdej Kanokmedhakul4, Anan Hiransalee3 and Ratsami Lekphrom5

Abstract

Drymycelia and culture filtrates from3 isolates (PW1, PW2 andKKU) of luminescentmushroom

(Neonothopanus nambi) were extracted in order to obtain bioactive compounds. Extraction using dry mycelium derived

bioactivecompoundpowdermorethanusingculturefiltratewithextractionefficiencyof8.73-12.90%.Theeffect

ofbioactivecompoundwasinvestigatedoninfectiouslarvae(J2)ofroot-knotnematode(Meloidogyne incognita) in

laboratory.Theresultshowedthatbioactivecompoundatconcentration500mg/lcaused100%mortalityofJ2in1

min.Concentrationsof100and50mg/laffectedJ2causingsignificantlymortalitiesof100%in30minand48hr,

respectively.EffectofthisbioactivecompoundonJ2wasalsoconfirmedinscreenedhouseexperimentandfoundthat

concentrationsof100and500mg/lsuppressedevidentlyJ2withoutroot-knotsymptomontomatoplants.Root-knot

score1wasdetectableontomatoplantstreatedwiththecompoundof10and50mg/l.However,itwassignificantly

(P<0.05)fromcontroltreatment,whichexhibitedroot-knotscore4.Thisstudysuggeststheefficiencyandpotential

of the application of bioactive compound from N. nambiforcontrolroot-knotnematode.

Keywords: Bioactivecompound,extraction,luminescentmushroom,root-knotnematode,tomato

1,5 Doctorate student, 2,4Associate Professor, 3Assistant Professor1,2,3 Center for Agricultural Biotechnology (AG-BIO/PERDO-CHE) and Agricultural Biotechnology Research Center for Sustainable Agriculture,

Khon Kaen University1,2,3 Department of Plant Science and Agricultural Resources, Faculty of Agriculture, Khon Kaen University, Khon Kaen, 40002, Thailand4,5 Department of Chemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand* Corresponding author, e-mail:[email protected]


and its Effect on Root-Knot Nematode (Meloidogyne incognita)

KKU Res J 15 (78) : August 2010

Introduction

It was reported that the luminescence

mushroomwasfoundinthePlantgeneticConservation

ProjectunderRoyalInitiationbyHerRoyalHighness

PrincessMahaChakri Sirindhorn at Kok Putaka,

WiangKaoDistrict,KhonKaenProvince.(Saksirirat

etal.,2003).Thisluminescentmushroomwassimilar

to the oyster mushroom by morphological characters.

Themushroomwaspoisonousandfoundintogroup,

each ofwhich consisted of 4-5 basidiocarps,which

normallygrewonlogsordeadwood.Duringdaytime,

themushroomhad gills,white cap, and short stipe.

During nighttime, especially during the dark night

orinthedarkroom,themushroomwasluminous.It

emits yellowish green light around the gills and stipe

thatvisiblewithinthefarrangearound10-20meters.

Initially,thestudywasclassifiedbyusingnucleotide

sequencesfromtheinternaltranscribespacer(ITS)of

rRNAgene.ItwascomparedtotheGenBankdatabase

atthattime.Later,themushroomlookedsimilartothe

onefoundinKokPutakawasfoundinthecampusof

KhonKaenUniversity.Duringdaytime,themushroom

waswhitecolor.Duringnighttime,itwasluminescent

withgreenandyellowcolors.Thenucleotidesequence

basesof ITS1-5.8S-ITS2of rRNA gene was studied

andcomparedtosequencedataofGenBankdatabase.

It was revealed that the DNA sequences were the same

asDNAsequencesofamushroom,Omphalotus sp.,

basedonavailabledatabaseat that time.Therefore,

it was previously reported that was the luminescent

mushroom in Omphalotus sp., found inKokPutaka,

KhonKaen.(Saksiriratetal.,2001;2004).Afterward,

Bua-art (2007) studied on the identification of this

luminescentmushroomfoundinKokPutaka(isolates

PW1andPW2)andthemushroomfoundinKhonKaen

Universitycampus(isolateKKU).Usingthenucleotide

sequencesofITS1-5.8S-ITS2regionofrRNAgene,it

wasfoundthatall3isolateshadthesamebasesequences

as nucleotides of Neonothopanus nambi Speg. The

identitywas 94%,when comparing the appearance,

color,andthemorphologicaldatafromtheexpertsin

luminescencemushroom(Prof.Dr.RoyWatling,Kew

Garden,Surley,UnitedKingdom,Dr.MatinKirchmair,

Institute ofMicrobiology,University of Innsbruck,

Innsbruck,Austria andDr. J.M.Monclavo,Centre

forBiodiversityandConservationBiology,Toronto,

Canada;Personalcontact).Itwasconcludedthatall3

isolatesofluminescentmushroomswerescientifically

named as Neonothopanus nambiSpeg.

Inothercountries,theystudiedtheadvantage

andexploitationofluminescentmushroom.Thebioactive

compound from luminescent mushroom was used

to biologically control plant diseases, especially to

control root-knot nematode (Meloidogyne incognita

Chitwood),which caused damage to the economic

crops over 3,000 species in temperate and tropical

areas. Engler et al. (1998) reported that secondary

metabolitewasisolatedfromculturefiltrateofluminescent

mushroom,Omphalotusolearius,inYMGmedium.The

mushroom O. oleariussecretedabioactivecompound,

omphalotin affecting the growth and development of

theroot-knotnematode(M. incognita).Later,Buchel

etal.(1998),identifiedthestructureofthatbioactive

compound from the luminescent mushroom O. olearius

usingSpectroscopy technique (1H and 13C Nuclear

MagneticResonanceSpectroscopy,1Hand13C-NMR)

andknownthat thebioactivecompoundaffectedthe

root-knot nematodewas omphalotin. There were

severalderivativesofomphalotins,suchasomphalotin

A,B,C,andD.Thesebioactivecompoundsinhibited

the nervous system and could be applied in controlling

theroot-knotnematode.



InThailand,therewasastudyontheeffectiveness

ofbioactivecompoundfromculturefiltratesofthe2

isolates,PW1,PW2ofluminescentmushroom,found

inKokPhutaka,KhonKaenandanotherisolatefound

inKhonKaenUniversity(KKU).Culturefiltratefrom

KKUisolatecausedthemortalityofroot-knotnematode

and reduced root-knot symptom in tomato 22.50%

significantlydifferentfromnouseofculturefiltrate.This

showed evidently that the luminescent mushroom can be

usedtocontroltheroot-knotnematode(Bua-art,2003).

Theinitialextractionandisolationofbioactivecompound

was done by using Thin Layer Chromatography

(TLC).An interesting bioactive compound was found

onseverallayersonTLC.Exampleswerethesubstances

inpulvinicacidgroup,thedifferentialofvariegatic,or

xerocomicacid.ThebioactivecompoundwithRf value

of0.41affectedthehighestmortalityofJ2larvaeofthe

root-knotnematode.Infact,PW1isolatehadthehighest

effectonthemortalityofJ2atupto100%(Bua-art,

2007).Nevertheless,thebioactivecompoundwithRf

valueof0.41hasnotbeenextractedthelargeamount,

identified and determined the chemical property of

thiscompound,aswellasthequalityofthepoisonous

property to the root-knot nematode. Therefore, the

purpose of this research was to extract the bioactive

compound from the luminescent mushroom N. nambi and

to examine the effect of bioactive compound against

root-knotnematode.Thiscanbeusedanddeveloped

furthertocontrolthepestwithabiologicalcontrol.This

wasthebeneficialwayfromthecountry’sbiological

resources, topreservetheenvironment,aswellas to

reduce the usage of pesticides.

Materials and Methods

1. The origin of the luminescence mushroom

(Neonothopanus nambi)

The 3 isolates of luminescentmushroom

(N. nimbi)wereused.TheywerePW1andPW2isolates

fromKokPhutaka,WiangKaoDistrict,KhonKaen

Province;andKKUisolatefoundintheareaofKhon

KaenUniversity.Themushroomhypheweregrownin

potatodextroseagar(PDA)orinsterilesorghumgrains.

Themushroomwasstoredat27°Cinordertobeused

conveniently in the study.

2. The extraction of the bioactive compounds from

the luminescent mushroom (Neonothopanus nambi)

ThreeisolatesofN. nambi were cultured in

PDAatthetemperatureof28±2°Cfor7days.Then,

the mushroom was grown in liquid culture malt extract

broth(MEB).Thebioactivecompoundwasextracted

biologically.Twoextractionmethodswereused, the

extractionmethod1usingculturefiltrate,andextraction

method2 usingdrymycelia as extractionmaterials.

Corkborerwiththeradiusof0.8centimeterwasused

to pierce agar with mycelium of luminescent mushroom.

Threepiecesofagarplugswereputintotheflaskwith

50milliliteroftheliquidmedium(MEB).ForType2,5

piecesofjellywereputintotheflaskwith50milliliters

oftheliquidfood(MEB).Themushroomculturewas

incubatedinthedarkroomwithoutshaking2hoursof

lightperdayat25°Cfor30days.Themyceliawere

filteredusingWhatmanfilterpaperNo.1tocollectthe

culturefiltrates.Thecollectedmyceliaweredriedinthe

ovenat45°Cfor3daysforfurtherextraction.




Theculturefiltratewasextractedusingethyl

acetate (EtOAc)3 times.Theextractedsolutionwas

collected and evaporated under pressure using the

rotary evaporator. The crude ethyl acetate (EtOAc)

was obtained.The crudeEtOAcwasweighted and

storedinthecontainer.Theweightofdrymyceliafrom

culturemediumwas recorded.Thedrymyceliawas

groundedusingtheblender.Theextractionforbioactive

compound was performed in the same manner as

describedpreviously.Thecrudeextractfromthedry

myceliaandtheculturefiltrateweretestedtodetermine

the effect of the bioactive compound on the infectious

larvae(J2)ofroot-knotnematode(M. incognita).To

crystallize the bioactive compound, the crude ethyl

acetate was separated using column chromatography.

SilicagelwasusedasstationaryphaseandEtOAc:hexane

assolvent.TheconcentrationofEtOAcwascontinuously

increased starting from 70%EtOAc:hexane. The

solution was collected together with the solvent as

fraction:100milliliterswerecollectedeachtime.Pole

strengthofthesolventwasincreasedupto100%EtOAc,

and then stopped. Each fraction was examined using thin

layerchromatography(TLC)inordertocombinethe

samefractiontogether.ThefractionswithspotsonTLC

were isolated for purification using chromatography

and crystallization until the extracted solution was

crystallized into powder.

3. Test of bioactive compound from luminescent

mushroom (Neonothopanus nambi) with root-knot

nematode (Meloidogyne incognita)

Laboratory Experiment

J2 larvae of the root-knot nematodewere

preparedbyadoptingtheprocessfromSomasri(2001).

Tomatorootswiththeroot-knotsymptomwerecleaned.

Clearly brown egg mass were collected using the forceps.

TheywerestoredinPVCpipewith3centimetersradius

and1centimeterheight.Thepipewassealedwithafine

netnyloncloth.Itwasimmersedin0.5%ofsodium

hypochlorite(NaOCl)for3minutes.Afterward,thepipe

wasrinsedwithsterilizeddistilledwater3times,for5

minuteseachtime.Thepipewasplacedonthecone

with 15-centimeterswidth.The cone had the rubber

tubeconnectedattheendwithclamp.Thewaterwas

poured just to the endof the pipe.After 2 days, J2

wouldhatchoutoftheeggs.Theycongregatedatthe

endoftherubbertubereadytobetested.Thepoeder

of bioactive compound from luminescence mushroom

was dissolved in dimethylsulfoxid (DMSO) at 50%

concentration with sterilized distilled water. Bioactive

compoundwaspreparedatconcentrationof0,10,50,

100,and500milligramperliter(mg/l)forfurthertest.

The effectiveness of bioactive compound

fromluminescentmushroomtotheroot-knotnematode

(M. incognita)wastestedinthelaboratory.TenJ2larvae

ofroot-knotnematodeweretestedintheholeofplastic

testingtray.Thereweretotalof24holesfilledwith300

µlofbioactivecompoundatconcentrationof0,10,50,

100,and500mg/lofeachhole.Thetraywasincubated

at25°C.Completelyrandomizeddesign(CRD)was

usedandconsistedof5treatments(eachconcentration

level of bioactive compound as treatment) and 4

replicates.Themortalityrateof theJ2wasobserved

byusingstereomicroscopeevery3hoursaftertreated

the J2with bioactive compound from luminescent

mushroom.

Greenhouse Experiment

J2nematodestestedwithbioactivecompound

fromluminescencemushroomatconcentrationof0,10,

50,100,and500mg/linthelaboratorywereverifiedon

the effectiveness of bioactive compound on prevention

ofroot-knotdiseaseinthegreenhouse.TenofJ2were



usedperconcentrationlevelfor3replicates.Theywere

immersedinbioactivecompoundatconcentrationof0,

10,50,100,and500mg/l.Thesolutionwasinfestedin

thesoilaroundtherootofthe21day-oldtomatoplants

(Sidavariety,PASeedsCompany)grownintheplastic

potswith15centimetersdiameter.Thecrudeextractof

bioactivecompoundof400µlwasusedforeachpotand

for each concentration. Completely randomized design

(CRD)consistedof6treatmentsand3replicateswas

used. After pouring the bioactive compound solution

with J2 for 7 days, tomatoeswere derivedurea and

irrigated normally. The plantswere inspected for

root-knot percentage after pouring the bioactive

compoundsolutionswithJ2for30daysaccordingto

thescoresfromSontirat(1998);0meantnoroot-knot

atall,1=1-25%ofroot-knot,2=26-50%ofroot-knot,

3= 51-75%of root-knot,whereas 4= 76-100%of

root-knot (highest level).The root-knot percentages

were analyzed statistically the variance and compared

theaveragevaluesusingDuncan’smultiplerangetest

(DMRT).

Results

1. The extraction of bioactive compound from

luminescence mushroom Neonothopanus nambi

Bioactive compound was obtained very little

byextractionmethod1.Usingthedrymyceliaprovided

moreamountofbioactivecompound(Table1).Total

drymyceliaweightof69.13gramsoffromisolatePW1

extractedusingEtOAc,wasachievedin15.77gramsof

crudecompound(22.81%ofextractionefficiencyw/w).

Thebioactivecompoundwasextracted from the2.5

litersofculturefiltrateusingEtOAc,resultingin4.63

gramsofcrudebioactivecompound(0.19%ofextraction

efficiency v/w).On the other hand, 105 grams of

bioactive compound was obtained from the dry mycelia

fromisolatePW2incrudecompoundweightof20.74

grams(19.75%ofextractionefficiencyw/w).However,

using10litersofculturefiltrate,itprovided16.75grams

of crude bioactive compound (0.17%of extraction

efficiencyv/w). ForKKU isolate, using 98.9 grams

of drymycelia, the bioactive compoundwas gained

withcrudecompoundweightof18.2grams(18.40%

ofextractionefficiencyw/w).Whenusing3.3litersof

culturefiltratetobeextractedbyextractionmethod2,

itprovided8.42gramsofcrudebioactivecompound

(0.26%ofextractionefficiencyv/w).Amongisolates

tested, the PW1 and PW2 gave rise to gain high

extraction efficiency 12.29-12.63%, as shown in

Table1.




Table 1. Comparisononthebioactivecompoundextractionfromdrymyceliaandculturefiltrateofluminescent

mushroom Neonothopanus nambi.

Extracted from the dry mycelia

Isolate Dry Crude Efficiency Bioactivecompound Efficiencyofextraction

weight (g) compound of extraction (%) powder (g) to powder (%)

(g)

PW1 69.13 15.77 22.81 8.73 12.63

PW2 105 20.74 19.75 12.90 12.29

KKU 98.9 18.20 18.40 10.62 10.74

Extractedfromtheculturefiltrate

Isolate Volume(l) Crude Efficiencyof Bioactivecompound Efficiencyofextraction

compound extraction (%) powder (g) to powder (%)

(g)

PW1 2.5 4.63 0.19 4.14 0.17

PW2 10 16.75 0.17 8.18 0.08

KKU 3.3 8.42 0.26 5.58 0.18

Column chromatography was used to purify

the crude bioactive compound and to crystallize the

solution.Crude compound extracted from3 isolates

of luminescent mushroom (N. nambi) was poured

through the column chromatography with reduced

pressureandcrystallizedusing20%MeOH:EtOAc.The

powderofbioactivecompoundtype1waslightyellow

color.Whensubjectedto theTLC, itwasfoundthat

this compound had Rf valueof0.46.Therewas8.73

gramsofPW1isolate(fromdrymycelia),4.14grams

(fromculturefiltrate)from12.90gramsofPW2isolate

(fromdrymycelia),8.18grams(fromculturefiltrate)

from10.62gramsofKKUisolate(fromdrymycelia),

and5.85grams(fromculturefiltrate).Inaddition,the

bioactivecompoundtype2wasachievedfromonlythe

isolatePW2,whichwasalsoyellowpowder,had Rf =

0.42whentestedonTLC(Figure1).



2.Efficiencyofbioactivecompoundextractedfrom

luminescent mushroom (Neonothopanus nambi)

against root-knot nematode (Meloidogyne incognita)

Laboratory Experiment

Bioactive compound from luminescent

mushroomwas tested in the laboratory against J2

larvaeoftheroot-knotnematode.Itrevealedthat,at500

mg/lconcentration,themortalityrateofJ2was100%

within1minute,followedby100mg/lconcentration

whichyieldedmortalityrateofJ2at100%within30

minutes.Attheconcentrationsof10and50mg/l,the

compoundcausedthemortalityofJ2within48hours.

Thiswassignificantlydifferentwithcontroltreatment

(J2inDMSO50%),whichindicatedthattherewasno

effectonthemortalityofJ2(mortalityrateat0percent).

Greenhouse Experiment

The effectiveness of bioactive compound

extracted from luminescent mushroom was tested in

the greenhouse in order to evaluate the mortality rate

ofJ2afterthetestinthelaboratory.Theresultshowed

that the bioactive compound at 100 and 500mg/l

concentrations were highly effective in controlling

theroot-knotnematodeinthetomatoes.Theroot-knot

roots were not found in the tomatoes of control

treatment having root-knot score at level 0 (no

root-knot).At the concentration of 10 and 50mg/l,

Figure 1. Bioactive compound strip from luminescence mushroom (Neonothopanus nambi);PW1,PW2,andKKU

isolatesexaminedby thin layerchromatography (TLC)using20%Methanol:EthylAcetate (1:1)per

volumeasthesolvent.1=bioactivecompoundtype1;2=bioactivecompoundtype2.




Table 2. Percentageoftheroot-knotintomatocausedbysoakinginfectiouslarvae(J2)oftheroot-knotnematode

(Meloidogyne incognita) in bioactive compound solutions from luminescent mushroom (Neonothopanus

nambi) at various concentration levels.

Bioactivecompound Percentageofroot-knot1 Root-Knot Score1

10mg/l 6.67c 1

50mg/l 11.67b 1

100mg/l 0d 0

500mg/l 0d 0

DMSO(50%) 82.33a 4

Distilledwater 82.67a 4

C.V. (%) 9.19

Meansfollowedbythesameletterarenotsignificantlydifferent(P>0.05,DMRT).10=noroot-knot;1=1-25%root-knot;2=26-50%root-knot;3=51-75%root-knot;4=76-100%root-knot.

(Sontirat,1998)

1-5 knotswere detected in tomato roots (level 1).

Thiswassignificantlydifferent(P<0.05)fromcontrol

treatment that having J2 alive. The root-knot

score level of this control treatmentwas at level 4

(76-100%), which showing stunt and root-knot

symptom.Thiswasclearlydifferentfromthetomatoes

treatedwith10-500mg/lofbioactivecompoundandJ2

ofroot-knotnematodes(Table2andFigure2).

Thisresearchresultindicatedthatextracting

the bioactive compound from N. nambi from the dry

mycelia gave more bioactive compound than extracting

fromtheculturefiltrate.Thisbioactivecompoundcaused

mortality of the root-knot nematode (M. incognita)

larvae.Besides,itpreventedthetomatoesfromroot-knot

disease. The test result in the laboratory and the

confirmationfromtheresultinthegreenhouseshowed

the effectiveness and nematicidal property of such

bioactivecompoundtocontroltheroot-knotnematode

in tomato.



Figure 2. Symptomofthetomatoplantsandrootsaftertreatedwiththebioactivecompoundfromtheluminescent

mushroom (Neonothopanus nambi) at 0, 10, 50, 100, and 500mg/l concentration, 400µl for each

concentration level. Ten J2 larvae of root-knot nematode (Meloidogyne incognita) were used per

concentrationlevelfor30days.

Conclusion and Discussion

Luminescent mushrooms (N. nambi) were

cultivated for the bioactive compound extraction.

Thedrymyceliagavemoreextractedcompoundthan

usingtheculturefiltrate.TheisolatePW1gave22.87

percentofcrudebioactivecompound,whichwasturned

into12.63percentofcompoundpowder,followedby

thoseofPW2andKKUisolates,respectively.Itwas

revealed that there was more bioactive compound from




luminescent mushroom (N. nambi) in the mycelia than

the secretion outside the mycelia.

Effectiveness of bioactive compound from

N. nambiwas testedwith J2 larvaeof the root-knot

nematode in the laboratory.Aconcentrationof 500

mg/lexhibitedthemortalityrateofJ2within1minute

aftertreated.Usingstereomicroscopy,J2wereinitially

movingveryquickly,andthenslowdownafterwards,

after about 1minute, it stoppedmoving.Therewas

nomovementorresponsefromJ2larvaewhenbeing

pushedusingthesmallbamboostick.Therunnerup

wastheconcentrationat100mg/l.Themortalityrate

ofJ2waswithin30minutes.Attheconcentrationof

10and50mg/l,themortalityrateofJ2waswithin48

hours, compared to control treatment (no bioactive

compound)thatnoeffectonthemortalityrateofJ2.

In order to verify that this bioactive compound was not

only nematostatic substance that caused the nematodes

tomomentarilystopmoving,thebioactivecompound

wastestedinthegreenhouseexperiment.Thiswasto

verifythemortalityofJ2inthelaboratoryandtheresult

recorded.TheJ2nematodesweresoakedinthebioactive

compoundsolutionattheconcentrationof100and500

mg/l.Thiscannotcausetheroot-knotsymptominthe

tomato plants. It meant that this bioactive compound

cancontrol the root-knotnematodes in the tomatoes

effectively.At theconcentrationsof10and50mg/l

ledthetomatoonlevel1ofhavingroot-knot.Thiswas

differentfromcontroltreatmentthathavinglivingJ2of

root-knotnematodes(0mg/l)withroot-knotscoreof

level4.Thesesuggestedthatthebioactivecompound

from luminescent mushroom caused the dead of

root-knotnematodesandpreventroot-knotdiseasein

tomatoes.

Thebioactive compounds that affected the

nematodes were reported that they can be extracted from

various plants and microorganisms. Aminobutyric acid

betaine,aminovalericacidbetaine,andglycinebetaine

wereusedinthetesting.Theywereextractedfromthe

brown seaweed (Ascophyllum nodosum),whichable

toreducethefemaleroot-knotnematodes.M. javanica

attacked Arabidopsis thaliana. (Wuetal.,1998).An

example of microorganism is Pochonia chlamydosporia

YMF1.00613thatinvadedeggsofroot-knotnematode,

M. incognita. TheP. chlamydosporia can produce

aurovertin I (A1) aurovertins E, F andD (A2-A4)

bioactive compounds. All 4 types of bioactive

compound were one factor that stopped the growth in

nematodes(Niuetal.,2010).Thebioactivecompound

in this aurovertin group was not only found in

P. chlamydosporia bacteria,but also inMetarhizium

anisopliae that caused the disease in insects and

secreted aurovertin D(1), F(2), G(3), and H(4)

bioactivecompounds(Azumietal.,2008).

ThereispreviousstudyfromAnke,Sterner

(1997),Buchel et al. (1998), on using the bioactive

compound from luminescent mushroom to control

the root-knot nematodes in plants.The luminescent

mushroom (Omphalotus sp.) can produce bioactive

compound,affectingmortalityofJ2ofroot-knotnematode.

Such bioactive compoundswere omphalotinA,B,

C,andD.Theextractionof thebioactivecompound

and the testing on the effectiveness of the bioactive

compound from the luminescent mushroom (N. nambi)

that were presented in this research were correlated

withtheresearchfromBua-art(2003).Thetestresult

ofsecondarymetabolitefromtheculturefiltrateof3

isolates of luminescentmushroom (PW1,PW2, and

KKU)againsttheroot-knotnematode(M. incognita)

was studied.That result indicated after pouring the

culturefiltrateofisolateKKUattheconcentrationof

10milliliterperpot,thenumberofroot-knotoftomato

plantshad22.50%reduction.Thiswasnotdifferent

fromusingnematicide, carbofuran.Thiswas similar



totheresearchfromSterneretal.(1997)reportedthat

mostluminescentmushroomcancontroltheroot-knot

nematode (M. incognita). This type ofmushroom

releasedomphalotintoxicsubstance.Meyeretal.(2004)

demonstrated that mushroom in family Omphalotaceae

can mostly create bioactive compound that affected

the root-knot nematode,which caused the root-knot

symptom.Forexample,beingabletocreateomphalotin,

the Omphalotus olearius was poisonous to the nervous

systemoftheroot-knotnematode(M. incognita).

The bioactive compound showing similar

effect from this luminescence mushroom (N. nambi) was

studiedbyBoehlendorfetal.(2004).AurisinAcanbe

extracted from the mushroom (Panus sp). It was not

luminescentmushroom, but affected several plant

pathogens,Pythium ultimum, Venturia inaequalis,

Plasmopara viticola, Puccinia graminis, and Phytopthora

infestans.However, therewas no report that such

compound affected the root-knot nematode.

Therefore, this research is the firstly in extracting

the bioactive compound from the luminescent

mushroom (N. nambi) and also including the result

of this nematicidal compound against the root-knot

nematode (M. incognita).Thispresentworkshowed

the way to use the bioactive compound (N. nambi)

to control the root-knot nematode. Furthermore,

the identificationof this compoundand its effect on

antagonisticmicroorganisms including other beneficial

organisms are studied in progress.

Acknowledgments

Wewould like to thank thePlantgenetic

ConservationProject underRoyal Initiation byHer

RoyalHighnessPrincessMahaChakriSirindhornfor

supportingtheresearch.Also,theCenterforAgricultural

Biotechnology(AG-BIO/PERDO-CHE)andAgricultural

Biotechnology Research Center for Sustainable

Agriculture,KhonKaenUniversitiy,fortheirsupport

on the funding and experimental equipments. The

appreciation included the Research group on Cultivation

andProductDevelopmentoftheWildSilkMothfor

Value addedCreation,KhonKaenUniversity for

allowing to use the equipments.

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