ENZYME NGOI BO NM Purpureocillium

lilacinum
PHN LP T TNH BNH PHC
L Th Thu Nhi*, L Th Mai Chm, V Thu Dng,
Phm Nguyn c Hong, Phm Hu Nhng
Trung tm Cng ngh Sinh hc Thnh ph H Ch Minh
*Email: thuynhi.sh@gmai.com
TM TT
Purpureocillium lilacinum c bit n nh mt tc nhn kim sot sinh hc ky
sinh tuyn trng gy hi thc vt. Purpureocillium lilacinumc th ky sinh tuyn trng nh kh
nng tit enzyme ngoi bo protease, chitinase, lipase ph v cu trc v trng cng nh biu
b tuyn trng ci. 67 chng nm Purpureocillium lilacinumphn lp rng v cc vn tiu
thuc tnh Bnh Phc c kho st enzyme ngoi bo bng phng php nui cy trn mi
trng thch. Kt qu, ng knh khun lc ca cc chng nm trn cc mi trng cm ng
ln nht sau 96 gi nhn nui, ng knh vng phn gii c cht cng ln nht cng thi
gian; tuy nhin, ty l gia ng knh vng phn gii trn ng knh khun lc ln nht 24
gi, v y nm tit enzyme mnh nht sau 24 gi nui cy. Cc chng nm c phn lp t
trng tiu c kh nng tit enzyme ngoi bo chitinase v protease cao hn cc chng nm c
phn lp rng. Trong , kh nng phn huy chitin, casein v tributyrin ca cc chng nm
c phn lp t vng rtiu b bnh tt hn so vi cc chng nm c phn lp t vng
r tiu kho mnh.
1. M U
Tuyn trng Meloidogyne sp. l tc nhn gy hi r cho nhiu loi cy nng nghi p
v cng nghi p nc ta. Trng v tuyn trng ci c bo v khi cc tc nhn ho hc v
sinh hc nh v trng v lp biu b thnh c th [4, 22]. V trng c cu to bi 3 lp chnh:
lp vetilline bn ngoi, lp chitin gia vlp glycolipid bn trong[3]. Lp vetilline cha
lipoprotein. Lp chitin gm chitin kt hp vi protein hnh thnh phc hp chitin protein
[5]. Nu lp chitin b ph huy th lp glycolipid s b nh hng [1].Thnh c th con ci c
cu to bi 3 lp: biu b, h b v c soma [5]. Biu b cha nhiu protein v chitin [13]. Cc vi
sinh vt xm nhp vo trng hay c th con ci thng qua v trng hay biu b thnh c th.
Enzyme c cho l nhn t quan trng gipnmxm nhpvo trng hay tuyn trng ci
[20].Purpureocillium lilacinum (Thom) Luangsa-ard, Houbraken, Hywel-Jones and Samson
(syn.Paecilomyces lilacinus)c kh nng ky sinh trng v con ci tuyn trng nh tit cc
enzyme protease, chitinase v lysozyme ph huy lp v trng hay biu b [6, 7, 8, 9,10,13,
19].Trong nghin cu ny, chng ti nh tnh cc enzyme ngoi bo protease, chitinase v
lipase ca 67 chng nm Purpureocillium lilacinum phn lp rng v t trng tiu tnh Bnh
Phc, hng ti ng dung trong kim sot tuyn trng gy hi cho cy tiu.

2. VT LIU V PHNG PHP NGHIN CU

2.1. Vt liu
17 chng nm P. lilacinum c phn lp t vn Quc gia B Gia Mp, 50 chng
nm Purpureocillium lilacinum c phn lp t cc vn tiu thuc tnh Bnh Phc (Bng 1)
v chng i chng P. lilacinum (NBRC; Nht Bn).
Mi trng nhn nui v gi ging l PDA (Sigma, Anh). Mi trng nh tnh
enzyme ngoi bo l Gause I, thay th c cht tinh bt bng chitin (Himedia, n ), casein
(Himedia, n ), v tributyrin (Sigma, Anh) vi thuc th tng ng l lugol (Merk, c),
TCA (Trichloroacetic acid, Unichem, Anh).
Bng 1. Danh sch cc chng P. lilacinum dng trong nghin cu
NGUN GC PHN LP
Vn Quc gia B Gia
Mp

Cc vn
tiu thuc
tnh Bnh
Phc

TN CHNG
BN1, BN2, BN3, BN5, BLO1, BLO3, BLO4, BHG5, BHG6,
BG1, BG3, BGG2, BGG4, BGG5, BGG6, BGG7, BGG9

Cy tiu
khe mnh

LN 1.3, LN 2.3, LN 4.2, HQ 1.3, HQ 5.3, HQ 6.3, HQ 7.3, BD

2.3, BD 3.3, BD 4.3, BD 5.3, BD 7.3, BGM1.1, BGM2.3,
BGM3.3, BGM5.2, BGM6.1

Cy tiu b
bnh

LN 1.2, LN 2.1, LN 3.2, LN 4.1, LN 4.3, LN 5.2, LN 6.1, LN

7.1, LN 7.2, HQ 1.2, HQ 3.1, HQ 3.4, HQ 5.1, HQ 6.1, HQ 6.2,
HQ 7.1, HQ 7.2, BD 2.1, BD 2.2, BD 3.2, BD 6.1, BD 7.1, BD
7.2, BGM1.3, BGM2.1, BGM2.2, BGM3.1, BGM4.1, BGM4.2,
BGM5.1, BMG6.2, BGM6.3, BGM7.1

2.2. Phng php nghin cu

2.2.1.

nh tnh enzyme ngoi bo

Cc enzyme ngoi bo ca nm cn nh tnh l chitinase, protease v lipase.
Huyn ph chitin bng HCl m c [19], huyn ph casein bng m phosphat (pH 7,6) [2].
Chun b mi trng cm ng tng hp enzyme ngoi bo nm v phn phi u ra a Petri.
Dng khoan thch ng knh 5 mm thu mu cy v chuyn sang gia a Petri cha mi trng
cm ng, 25 40C. Nhum thuc th lugol, TCAv o ng knh khun lc, ng knh
vng phn gii c cht sau khi kt thc th nghim [17, 14, 16, 20].
Ch tiu theo di: ng knh tn nm (d, cm), ng knh vng phn gii c cht
(D, cm) v t l D/d sau 24, 48, 72 v 96 gi nui cy.

2.2.2.

Phn tch thng k

Th nghim nh tnh h ezyme ngoi bo ca 67 chng nm c b tr theo kiu
hon ton ngu nhin vi 3 ln lp li. S liu ng knh khun lc v ng knh vng phn
gii c o trc tip, tnh t l D/d bng chng trnh excel. S dung phn mm SAS 9.1
Portable phn tch ANOVA, tnh gi tr trung bnh v so snh trung bnh qua php th Duncan
vi mc y ngha 5% .

3. KT QU
C s khc bit rt c y ngha v mt thng k v kh nng tit enzyme ngoi bo
ca cc chng nm phn lp cc a im khc nhau. Kh nng phn hy c cht cng khc
nhau rt c y ngha v mt thng k theo thi gian. Kh nng phn hy c cht cao nht l 96
gi sau cy v gim dn khi thi gian nui cy gim. ng knh tn nm biu th cho s pht
trin ca nm trn mi trng c c cht cm ng v ng knh tn nm ca tt c cc chng
u ln nht sau 96 gi nhn nui. Tuy nhin, t l gia ng knh vng phn gii c cht v
ng knh tn nm biu th cho thi gian phn hy c cht mnh nht cng khc nhau rt c y
ngha v mt thng k theo thi gian v gia cc chng nm khc nhau. T l D/d cao nht l
24 gi v gim dn khi thi gian nui cy tng ln.
3.1.So snh kh nng tit enzyme ngoi bo ca cc chng nm c phn lp rng v
t trng tiu
3.1.1. Enzyme chitinase
3.500
3.000
2.130
2.500
1.739 1.761
2.000
1.500
1.000
0.500 Nm phn lp t VQG B Gia Mp
0.000

3.097 3.141 3.157

3.011 2.795 3.192

Nm phn lp t cc vn tiu Bnh Phc

Purpureocillium lilacinum

Hnh 1. Biu so snh ng knh tn nm, ng knh vng phn gii chitin v ty l gia ng knh
vng phn gii trn ng knh tn nm ca cc chng phn l p cc vng khc nhau v chng i chng
Purpureocillium lilacinum nui cy trn mi trng cha chitin.

Thi gian u nui cy (24 gi), cc chng nm phn lp t rng c c kh nng

phn huy chitin mnh hn so vi cc chng nm c phn lp t trng tiu nn lm cho t l
D/d cng cao hn. Tuy nhin, sau 96 gi, kh nng ny li thay i, theo , ng knh vng
phn gii chitin ca cc chng nm phn lp t trng tiu l cao hn. Xt v kh nng pht
trin trn mi trng c c cht chitin, thy rng cc chng nm phn lp t t trng tiu c
kh nng pht trin tt hn so vi cc chng nm c phn lp t rng sau 96 gi nui cy.
Khi tnh trung bnh th ng knh tn nm, ng knh vng phn gii c cht v ty l gia D/d
ca cc chng nm phn lp nh hn so vi chng i chng; tuy nhin, c mt s chng phn
lp c u th v cc ch tiu ny hn so vi chng i chng, iu ny c th hin qua
lch chun th hin trn hnh 1.

Hnh 2. ng knh vng phn gii chitin (vng trong) ca cc chng nm phn lp t t rng (a) v t
trng tiu (b) sau 96 gi nui cy.

3.1.2. Enzyme protease

ng knh tn nm i din cho kh nng mc v pht trin trn mi trng cm
ng, do , ng knh tn nm cng ln th chng t nm cng c kh nng pht trin mnh
trn mi trng . Kh nng pht trin trn mi trng c casein ca cc chng c phn lp
t rng tt hn do c ng knh tn nm ln hn so vi cc chng c phn lp t trng
tiu. Sau 24 gi nui cy, hu ht cc chng nm c phn lp t rng c th tit enzyme
protease mnh hn nn lm cho t l D/d cao hn, tuy nhin, khi thi gian nui cy tng dn th
cc chng nm c phn lp t trng tiu li c u th v kh nng tit enzyme ngoi bo
hn (ng knh vng phn gii casein ca cc chng phn lp t t trng tiu l 2.396 cm, cao
hn so vi cc chng nm phn lp t t rng).
3.500
3.000
2.060 1.943 2.130
2.500
2.000
1.500
1.000
0.500 Nm phn lp t VQG B Gia Mp
0.000

3.192

3.157
2.370 2.396

2.351 2.140

Nm phn lp t cc vn tiu Bnh Phc

Purpureocillium lilacinum

Hnh 3. Biu so snh ng knh tn nm, ng knh vng phn gii casein v ty l gia ng knh
vng phn gii trn ng knh tn nm ca cc chng phn l p cc vng khc nhau v chng i chng
Purpureocillium lilacinum nui cy trn mi trng cha casein.

Hnh 4. ng knh vng phn gii casein (vng trong) ca cc chng nm c phn lp t rng (a)
v t trng tiu (b) sau 96 gi nui cy.

3.1.3. Enzyme lipase

3.500
3.000
2.500
2.000
1.482 1.538 1.506
1.500
1.000
Nm phn lp t VQG B Gia Mp
0.500
0.000

2.718 2.583 2.893

3.233
2.920 3.029

Nm phn lp t cc vn tiu Bnh Phc

Purpureocillium lilacinum

Hnh 5. Biu so snh ng knh tn nm, ng knh vng phn gii tributyrin v ty l gia ng
knh vng phn gii trn ng knh tn nm ca cc chng phn l p cc vng khc nhau v chng i
chng Purpureocillium lilacinum nui cy trn mi trng cha tributyrin.

Xt v kh nng mc v pht trin trn mi trng c c cht tributyrin, chng ti

thy rng cc chng nm c phn lp t trng tiu c u th hn, iu ny th th hin qua
ng knh tn nm trung bnh ca cc chng ny cao hn hu ht cc mc thi gian kho st.
Tuy nhin, xt v kh nng phn huy c cht tributyrin th cc chng nm c phn lp t
rng c u th hn hu ht cc mc thi gian kho st, iu ny th hin qua ng knh vng
phn gii tributyrin. Cc chng nm phn lp t rng c ng knh vng phn gii sau 96
gi l 2,718 cm, trong khi ng knh vng phn gii c cht ca cc chng nm phn lp
t trng tiu l 2,583 cm. Do c kh nng phn huy tributyrin mnh hn so vi cc chng nm
c phn lp t trng tiu nn t l D/d ca hu ht cc chng nm phn lp t rng cao
hn hu ht thi gian kho st; tuy nhin, sau 24 gi nui cy th cc chng nm c phn
lp t trng tiu c tc phn huy c cht mnh hn (t l D/d sau 24 gi ca cc chng
nm phn lp t trng tiu l 3,029 v t rng ch l 2,92).

Hnh 6. ng knh vng phn gii tributyrin (vng trong) ca cc chng nm phn lp rng (a) v t
trng tiu (b) sau 96 gi nui cy.

3.2. So snh kh nng tit enzyme ngoi bo ca cc chng nm c phn lp vng r

tiu bnh v kho mnh
3.2.1. Enzyme chitinase
3.500
3.000
2.500
2.000
1.500
1.000
0.500
0.000

3.170 3.075 3.157

2.742 2.914

3.192

2.130
1.751 1.785
Nm phn lp t t xung quanh gc tiu b bnh

Nm phn lp t t xung quanh gc tiu kho mnh

Purpureocillium lilacinum

Hnh 7. Biu so snh ng knh tn nm, ng knh vng phn gii v ty l gia ng knh vng
phn gii trn ng knh tn nm ca cc chng phn l p t vng r tiu vi mc bnh khc nhau
v chng i chng Purpureocillium lilacinum nui cy trn mi trng cha chitin.

Khi nui cy cc chng nm trn mi trng cha c cht chitin, chng ti thy
rng cc chng nm phn lp t t vng r tiu kho mnh c kh nng pht trin nhanh hn
cc chng nm cn li (ng knh tn nm t c sau 96 gi nui cy l 1,785 cm). Lc u
(sau 24 gi nui cy), cc chng nm ny cng tit enzyme rt mnh nn lm cho t l D/d t
gi tr cao nht (2,914). Tuy nhin, khi thi gian nui cy tng dn th cc chng nm phn lp
t t vng r tiu b bnh tit enzyme c hot tnh mnh hn cc chng nm cn li nn lm
cho ng knh vng phn gii c cht chitin ln hn (hnh 7).

Hnh 8. ng knh vng phn gii chitin (vng trong) ca cc chng nm phn lp t vng r tiu b
bnh (a) v khe mnh (b) sau 96 gi nui cy.

3.2.2. Enzyme protease

3.500
3.000
2.500
2.000
1.500
1.000
0.500
0.000

2.880

2.967

2.403 2.379

2.280
2.156 2.103
1.917 2.002
Nm phn lp t t xung quanh gc tiu b bnh

Nm phn lp t t xung quanh gc tiu kho mnh

Purpureocillium lilacinum

Hnh 9. Biu so snh ng knh tn nm, ng knh vng phn gii v ty l gia ng knh vng
phn gii trn ng knh tn nm ca cc chng phn l p t vng r tiu vi mc bnh khc nhau
v chng i chng Purpureocillium lilacinum nui cy trn mi trng cha casein.

Cc chng nm phn lp v i chng c kh nng pht trin nhanh hn trn mi

trng c c cht casein, tuy nhin hot tnh enzyme protease t cc chng ny li yu hn so
vi enzyme chitinase. Cc chng nm phn lp t t vng r tiu b bnh c th tit enzyme
protease vi hot tnh mnh hncc chng phn lp t t vng r tiu khe mnh sau 24 gi
nhn nui.Sau 96 gi nui cy, ng knh tn nm ca cc chng phn lp t t vng r tiu
kho mnh ln hn (2,002 cm); iu ny th hin rng cc chng nm phn lp t t vng r
tiu kho mnh c tc pht trin nhanh hn trn mi trng c casein so vi cc chng nm
cn li. Tuy nhin, hot tnh enzyme ngoi bo protease cc chng nm phn lp t t vng
r tiu b bnh mnh hn so vi cc chng phn lp t t vng r tiu kho mnh, iu ny th
hin qua ng knh vng phn gii c cht casein sau 96 gi nui cy (hnh 9).

Hnh 10. ng knh vng phn gii casein (vng trong) ca cc chng nm vng r tiu b bnh (a) v
khe mnh (b) sau 96 gi nui cy.

3.2.3. Enzyme lipase

3.500
3.000
2.500
2.000
1.500
1.000
0.500
0.000

2.594 2.558

2.893

3.040 3.003 3.233

1.535 1.547 1.506

Nm phn lp t t xung quanh gc tiu b bnh

Nm phn lp t t xung quanh gc tiu kho mnh

Purpureocillium lilacinum

Hnh 11. Biu so snh ng knh tn nm, ng knh vng phn gii v ty l gia ng knh vng
phn gii trn ng knh tn nm ca cc chng phn l p cc cy tiu vi mc bnh khc nhau v
chng i chng Purpureocillium lilacinum nui cy trn mi trng cha tributyrin.

Cc chng nm phn lp pht trin chm hn trn mi trng c c cht tributyrin

so vi casein v chitin. Sau 96 gi nui cy, ng knh tn nm trung bnh ca tt c cc chng
u nh hn 1,6 cm; trong , cc chng nm c phn lp t t vng r tiu khe mnh c
ng knh tn nm trung bnh ln hn. Tuy nhin, kh nng tit enzyme lipase c hot tnh
mnh hn thuc v cc chng nm c phn lp t vng r tiu b bnh, sau 24 gi nui cy
cc chng ny c ty l D/d l 3.040 v sau 96 gi th enzyme tit ra t nhm nm ny c kh
nng phn huy c cht tributyrin t ng knh vng phn gii trung bnh 2,594 cm (hnh 11).

Hnh 12. ng knh vng phn gii tributyrin (vng trong) ca cc chng nm vng r tiu b bnh (a)
v khe mnh (b) sau 96 gi nui cy.

4. THO LUN
Kh nng pht trin trn mi trng c c cht cm ng v phn hy c cht thp
nht l 24 gi sau cy v tng dn khi thi gian nui cy tng ln. iu ny c th l do thi
gian nui cy cng lu th nm cng thch nghi dn vi c cht cm ng c trong mi trng v
chng phi phn hy c cht thnh cc cht n gin hn dinh dng nn ng knh tn
nm v ng knh vng phn gii c cht cng lc cng ln. Tuy nhin, t l gia ng knh
vng phn gii v ng knh tn nmcao nht l 24 gi v gim dn khi thi gian nui cy
tng ln. iu ny c ngha l cc chng nm tit enzyme c hot tnh mnh nht sau 24 gi
nui cy. Nguyn nhn c th l do khi mi tip xc vi mi trng c c cht cm ng, nm
phi tit ra mt lng enzyme phn hy c cht lm ngun dinh dng nui chng trong
thi gian u m thi gian ny chng mi tip xc vi mi trng mi nn tn nm khng pht
trin, v vy lm cho t l D/d ln nht. Theo thi gian, nm thch nghi dn vi mi trng
nn chng pht trin nhanh lm cho ng knh tn nm ln nhanh, trong khi hot tnh enzyme
yu hn so vi lc ban ulm cho t l D/d cng gim dn.
Tuyn trng xp hng th hai sau cn trng v s lng loi v ng u v s
lng c th trong gii ng vt. Tuyn trng hin din khp mi ni: trong t canh tc, trong
t rng, trong t trng c,. [23]. Vng r cy h tiu trng trn t canh tc cng hin din
nhiu loi tuyn trng ngoi ky sinh v ni ky sinh, trong Meloidogyne spp. ky sinh trn
100% mu r ca cc ging h tiu canh tc[22]. Do c s hin din thng xuyn ca tuyn
trng trong t canh tc nn lm cho hot tnh enzyme ngoi bo protease v chitinase ca cc
chng nm phn lp t vng ny mnh hn so vi cc chng nm phn lp t t rng.
i vi cc chng nm phn lp t t canh tc, hot tnh enzyme ngoi bo
chitinase, protease v lipase mnh nht t cc chng phn lp t gc tiu b bnh. iu ny c
th do s hin din ca tuyn trng trong t v r ca cy b bnh nhiu hn so vi gc tiu
kho mnh. Trnh Th Thu Thuy v cng s iu tra v thnh phn tuyn trng trong t

trng tiu pht hin c 29 loi tuyn trng ky sinh thc vt, trong ph bin nht l tuyn
trng thuc chi Meloidogyne. V s lng Meloidogyne spp. c lin quan ti ch s gy hi ca
tuyn trng trn r, cy b bnh vng l c phn trm nt sng r cao th s lng tuyn trng
trong r v t xung quanh cng nhiu[21]. Quan st r ca cc cy tiu b bnh th thy c
nhiu nt sng hn so vi cy tiu kho mnh. Qu trnh ky sinh trng v con ci tuyn trng
bi nm bt u bng vic tit enzyme enzyme ngoi bo ph hy lp v trng hay biu b
ca con ci[6, 7, 9, 13]. Enzyme chitinase v proteasel hai enzyme chnh v u tiu gip
P.lilacinum xm nhp vo c th con ci hay trng tuyn trng
M.javanica[1]. Do , cc chng nm c phn lp t cc gc tiu b tit enzyme ngoi bo
chitinase, protease v lipase c hot tnh mnh hn so vi cc chng nm phn lp t gc tiu
khe mnh.
Li cm n. ti c thc hin nh kinh ph thng nin ca Trung tm Cng ngh Sinh hc. Chng
ti xin chn thnh cm n TS. Dng Hoa X v cc anh ch phng Cng ngh Vi sinh Trung tm Cng
ngh Sinh hc gip chng ti hon thnh ti ny.M s ti: VS02/13 15

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ABSTRACT

EXTRACELLULAR ENZYMES OF Purpureocillium lilacinumSTRAINS

ISOLATED IN BINH PHUOC PROVINCE
L Th Thu Nhi*, L Th Mai Chm, V Thu Dng,
Phm Nguyn c Hong, Phm Hu Nhng
Bioteachnology center of Ho Chi Minh City
*Email: thuynhi.sh@gmail.com

Purpureocillium lilacinum is known as a biological control agent for plant parasitic nematodes.
Purpureocillium lilacinum infects nematodes by secreting extracellular protease, chitinase and
lipase to degrade nematode eggshell structure as well as cuticle structure of females. 67 strains
of Purpureocillium lilacinum were isolated from the forest soil and cultivated black pepper soil
in Binh Phuoc Province. The qualitative test of extracellular enzymes was productedof those
strains by substrate clear zone method. In results, the fungal colony diameter of those strains on
agar medium were the largest after 96 hours of incubation, formation of clearing zones around
the fungal growth were also the largest in the same time point. However, base on the rate

between diameter of clear zone and diameter of fungal colony, extracellular enzymes of them
were the best active after 24 hours of culture. The ability to secrete extracellular chitinase and
protease ofthe strainsfromcultivated black peppersoil were betterthan thosefrom the forest soil.
Among them, the ability to degrade chitin, casein and tributyrin of the strains isolated in
rhizospheral soil of disease black pepper plantation were better than isolates from rhizospheral
soil of healthy black pepper plantation.
Keywords:Purpureocillium lilacinum, extracellular enzyme, chitinase, protease, lipase.