Báo cáo " Đánh giá khả năng làm giảm dư lượng Sherpa, Regent và Pegasus của một số chủng vi khuẩn tuyển chọn" docx

TAP CHi KHOA HOC
VACONG
NGHE Tgp 47, s6
1,2009
Tr. 65-71
DANH GIA KHA NANG LAM GIAM DU LTONG SHERPA,
REGENT VA PEGASUS CUA MOT SO
CHONG VI KHUAN
TUYEN CHON
NGUYEN THI KIM CUC, LE
TH!
HCNG MINH, PHAM VIET CUCNG
1.
MO DAU
Hda chit bao ve thuc vat (HCBVTV) la ten ggi chung cho cac chat diet sau bg (pesticides),
diet cdn triing (insecticides), diet nam (fungicides) va diet cd (herbicides). Phan ldn hoa chat
BVTV thudc nhdm hiiru ca khd phan buy. HCBVTV da dugc sir dung d Viet Nam tir nhirng nam
40 vdi sd lugng ngay cang tang.
Theo cac bao cao gan day nhat ciing nhu cac ket qua dieu tra ciia Vien BVTV thi viec lam
dung va sir dung cac thudc BVTV cd do ddc cao, tham chi cac loai thudc ban che va thudc cam
vin dang dien ra. Tren rau hg thap tu, phat hien 17 boat chat trir sau trong do cd 6 thudc thudc
nhdm lan huu ca, 1 thudc Cacbamate, 5 thudc Pyrethroid, 2 thudc dieu boa sinh trudng, 1 hdn
hgp va 2 thuoc thudc cac nhdm khac. Hai trong sd 17 thudc thudc nhom doc I, 10 thudc thudc
nhom II, 1 thuoc nhom III va 4 thudc nhom IV. Cac thudc nay dugc sir dung phd bien nhat la,
Methamidophos (80,4%), sau dd dSn Cypermethrin (39,5%); CIDl-M (34,8%); Cartap (30,8%);
Triclorfol (28,3%) va Methylparathion (25,4%).
Tai Hdi nghj toan quoc ve cdng tac bao dam v? sinh an toan thuc pham dugc td chirc ngay
10/01/2007, Bd Ndng nghiep va Phat triSn Ndng thdn thira nhan mire do tdn du HCBVTV d rau
va qua la kha nghiem trgng va dang lo ngai. Rau cai dirng dau vdi ti le mau cd du lugng TBVTV
cao nhit nam 2004 la 63,9%. Tuy nhien, ti le mau cd du lugng thudc vugt mirc cho phep lai
dimg sau dau do (4,2 - 10,18% so vdi 10 - 27,5%). Cd den 70% sd mau rau an la dugc tim thay

cd du lugng Pyrethroid, sd cdn lai danh cho Fipronil, Dithiocarbamate, mdt sd loai lan hiru ca va
Carbendazim [Bao KH & DS, 5 (1931), 18/1/2007].
Trong thien nhien, vi sinh vat dat dugc eoi nhu mgt nhan td tich cue tham gia vao chu trinh
van chuyin cua cac nguyen td boa hgc va lam can bang trang thai tir nhien ciia mdi trudng. Rat
nhieu chi vi sinh vat da dugc phan lap va chimg minh cd kha nang phan buy cac boa chat ddc hai
do con ngudi thai vao mdi trudng nhu vi khuan thudc chi Flavobacterium (Sethunathan &
Yoshida 1973), Pseudomonas (Serdar et al. 1982), Agrobacterium, Bacillus (Home et al. 2002).
Viec khir ddc HCBVTV bing enzyme vi sinh vat trd thanh tam diem ciia rat nhieu nghien cim,
vi svr hieu qua va kinh te ciia phuang phap nay.
Myc tieu ciia nghien ciru nay la phan lap va tuyen chgn vi khuan cd kha nang lam giam du
lugng Pegasus, Sherpa hoac Regent sir dung trong Ndng nghiep.
2.
NGUYEN LIEU VA PHlTONG PHAP
2.1.
Nguyen lieu
65
Cac chung vi khuin dugc phan lap tir cac mau dat cd sir dung Pegasus, Sherpa hoac Regent
dk phdng trir benh hai cay
Mdi trudng phan lap (g/1): NaMo04.2H20 - 0,33ml tir dung djch pha sin (stock) 1%;
KH2PO4 - 4,8; K2HPO4'- 1,2; NH4 NO3 -1; MgS04.7H20 - 0,2; Ca(N03)2 - 0,4 ml tir stock
10%;
Fe(S04)3 - 10 ml tir stock 10%; pH trung tinh. Ngudn nang lugng va cacbon la HCBVTV
vdi nong do gip 2,5 -5 lin so vdi ndng do thudng dugc sir dung (2,5 mg/ml cho Regent va
Pegasus va 1,3 mg/ml cho Sherpa).
2.2.
Phu'o'ng phap
Cac chimg vi khuin dugc phan lap theo phuang phap lam giau: mau dat (0,5 g) dugc dua
vao mdi trudng tdi thik (MM) cd bo sung HCBVTV va nudi lie trong 48 gid d 30°C. Cay
chuyin 1 ml djch nudi ciy sang mdi trudng mdi va ti^p tuc nudi ukp trong 2 ngay. Pha bang
dich nudi ciy va ciy trai tren mdi trudng dac (MM±HCBVTV). Chgn cac khuan lac phat trien

tdt tren mdi trudng nay sau 24 gid nudi cay d 30°C.
- Phuang phap xac djnh ton du HCBVTV: LC/DAD-FL (cho Pegasus) va GC/EDC (cho
Sherpa va Regent) dugc thuc hien tai Nhdm Phan tich, Bd mdn Thudc, vien Bao ve Thuc vaL
3.
KET QUA VA THAO LUAN
3.1.Danh gia kha nang sinh trirdng cua vi khuSn tren MM vdi cac loai HCBVTV khac
nhau
Cac chiing vi khuin sau khi dugc lam giau va tuyen chgn rieng re tren mdi trudng tdi thieu
CO
bd sung Pegasus, Sherpa va Regent dugc kiem tra kha nang sinh trudng ddng thdi tren moi
trudng MM vdi cac loai HCBVTV khac nhau. Kha nang sinh trudng ciia cac chiing vi khuan
tuyen chgn sau 24 gid nudi cay dugc trinh bay tren bang 1.
Bdng
1.
Kha nang sir dung Pegasus, Sherpa va Regent ciia cac chiing vi khuan tuyen chgn
SdTT
1
0
3
4
5
6
7
8
9
10
11
12
Ten
chiing

PI
P2
P
RI
R2
R3
R4
R5
R6
R7
R8
R9
Regent
++
+-I-
++
-H-
++
++
-H-
++
++
++
-I-+
-1-4-
Pegasus
++
++
+-I-
-H-

-l-l-
++
++
++
+-I-
+-I-
++
-l~l-
Sherpa
-I-+
++
-I-+
++
+
-H-
++
+
+-I-
-1-
++
++
SoTT
13
14
15
16
17
18
19
20

21
22
23
24
Ten
chiing
RIO
Rll
SI
S2
S3
S4
S5
S6
S9
SIO
S12
S16
Regent
++
++
++
+
+
++
++
-1-
±
++
+

-H-
Pegasus
++
++
++
+
++
-1-
+
++
++
+
++
-H-
Sherpa
+
-i-i-
-I-+
+-I-
++
++
-I-+
-I-+
++
++
++
++
Ghi chu:
+ + .
sinh trudng tdt; +: sinh truang trung hinh.

66
Ket qua tren bang 1 cho thay cac chiing tuyen chgn mac dii dugc phan lap tren cac ngudn
cacbon khac nhau deu cd kha nang sinh trudng tdt tren mdi trudng tdi thieu cd bd sung cac loai
thudc BVTV khac nhau. Vi du: cac chiing dugc phan lap tren mdi trudng bd sung Pegasus (ki
hieu P), sinh trudng tdt tren mdi trudng tdi thieu bd sung Regent va Sherpa. Ngugc lai, cac
chimg phan lap tren mdi trudng vdi ngudn cacbon la Regent (ki hieu R) va Sherpa (ki hieu S)
cung cd the sinh trudng tdt tren mdi trudng cd Pegasus va Sherpa.
Da kiem tra anh hudng cua agar len sir sinh trudng ciia cac chiing tuyen chgn bang each
danh gia kha nang sinh trudng ciia cac ehiing nghien ciru trong mdi trudng long vdi ngudn
cacbon la HCBVTV. Ket qua nhan dugc cho thay, trong mdi trudng Idng, mdt sd chiing sinh
trudng kem ban so vdi khi cay tren mdi trudng dac (ket qua khdng cdng bd).
Bdn chiing SI, SI6, PI va RI sinh trudng tdt ca tren mdi trudng dac va mdi trudng long cd
bd sung mgt trong 3 loai HCBVTV tren da dugc chgn de danh gia boat tinh lam giam du lugng
Pegasus, Sherpa va Regent.
3.2.
Danh gia hoat tinh giam dir lircfng HCBVTV ciia mot so chiing vi khuan tuyen chon
Cac chiing vi khuan tuyen chgn dugc nudi trong mdi trudng tdi thieu bd sung HCBVTV.
Ndng do thudc BVTV bd sung vao mdi trudng dugc tang len gap 4 lan so vdi ndng do thudng
dugc sir dung ngoai ddng rudng. cu the Pegasus 3 mg/ml; Regent 0.5 mg/ml va Sherpa 1.24
mg/ml. Bang phuang phap phan tich LC/DAD-FL hoac GC/ECD, da xac djnh du lugng thudc
cdn lai trong mdi trudng sau 3 ngay nudi cay. Ket qua dugc trinh bay tren bang 2.
Bdng 2. Kha nang lam giam du lugng thudc BVTV trong mdi trudng nudi cay ciia
mdt sd chiing vi khuan tuyen chgn
STT
1
2
3
Ten chiing
S16
SI

PI
RI
DC
S16
SI
PI
RI
DC
S16
SI
PI
RI
DC
Ten du lugng
Diafcnthiuron
(Pegasus)
Cypermethrin
(Sherpa)
Fipronil
(Regent)
Ket qua (mg/l)
846,21
679,17
623,49
882,58
960,27
80,13
146,82
133,60
54,31

327,00
21,82
37,70
64,03
28,45
93,88
Phuang phap
LC/DAD-FL
GC/ECD
GC/ECD
% giam so vdi DC
11,88
29,27
35,07
8,09
75,50
55,10
59,14
83,39
76,76
59,84
31,80
69,70
67
Bang 2 cho thay cac chimg vi khuan nghien ciru da lam giam lugng Pegasus, Regent va
Sherpa trong mdi trudng sau 3 ngay nudi cay tir 8,09% din 83,39%. Dac biet, hai chiing S16 va
RI cd boat tinh cao ddi vdi Regent va Sherpa. Chiing SI6 lam giam 76,76% Regent va 75,5%
Sherpa so vdi doi chimg; trong mdi trudng nudi cay cd cbting RI, sau 3 ngay lugng Regent va
Sherpa giam so vdi doi chimg la 69,7% va 83,39% tuong img. Nhu vay, mac dii sir sinh trudng
cua cac chiing tuyen chgn tren mdi trudng dac cd bo sung HCBVTV tuong duang, nhung hoat

tinh sinh hgc ciia chiing cd su khac biet rd ret.
Bon chung vi khuin tuyen chgn mac dii sinh trudng tot trong mdi trudng cd Pegasus,
nhung chiing chi lam giam Pegasus dugc tir 8,09% den 35,07% so vdi ddi chung sau 3 ngay
nudi cay. Vi vay, mdt sd chiing vi khuan trong bd suu tap gidng cua phdng Vi sinh phan tir co
kha nang phan hiiy 2,4- dichlorophenoxyacetic acid da dugc sir dung de xac djnh kha nang loai
ddc Pegasus.
3.4.
Hoat tinh lam giam nong do Pegasus cua 3 chung vi khuan tuyen chon
Ba chiing vi khuin K5, NT64 va LT33 da dugc chgn de xac djnh kha nang loai Pegasus. Vi
khuin dugc nudi trong mdi trudng tdi thieu cd bd sung Pegasus ndng do 3 mg/ml. Sau 3 ngay
nudi ciy d 30°C, du lugng Pegasus trong mdi trudng dugc xac djnh bang phuang phap
LC/DAD-FL. Ket qua dugc trinh ifay tren bang 3.
Bdng
3.
Hoat tinh ciia 3 chiing vi khuan nghien ciru
Ten chiing
K5
NT64
LT33
DC
Ham luang
(mg/l)
362.10
301,52
397,48
953,62
% giam so vdi
DC
62,03
68,38

58,32
Kha nang sinh trudng
Pegasus
++
-H-
++
Regent
++
+
++
Sherpa
-
-
-
Ket qua nhan dugc cho thay 3 chiing vi khuan nghien ciru cd the sir dung de loai bd du
lugng Pegasus trong thuc te.
Da cd nhieu nghien ciru ciia cac tac gia khac tren the gidi thdng bao ve viec phan lap va sir
dung vi sinh vat de loai ddc HCBVTV. Smeijkal C. W. va cs (2001) da phan lap va tuyin chgn
cac chiing vi khuan phan hiiy hgp chat do hiru ca bang mdi trudng dac hieu (Loos). Cac tac gia
nhan thay mdt sd chiing vi khuan phan hiiy 2,4-D cung cd kha nang phan hiiy mdt so hgp chit
khac nhu MCPA, (R/S)-mecoprop, (R)-mecoprop (R/S)-2,4-DP, 2,4,5-T. 2,4-DB va MCPB.
N.
T. K. Ciic va cs (2000) phan lap cac chiing vi khuin phan hiiy Methyl parathion tren mdi
trudng tdi thieu vdi ngudn cacbon la Methyl parathion, cac chiing nay cung cd kha nang sinh
trudng tren mdi trudng tdi thieu vdi ngudn cacbon la cac chit trir sau khac nhu Lindan va
Kitazin. Ben c^nh dd, mdt sd chiing vi khuan phan hiiy thuoc trir cd phenoxyalkanoic acid lai co
ca chat ban che. Dugc biet sd lugng va vj tri thay
Xhk
ciia do anh hudng d^n kha nang phan hiiy
ciia rat nhieu vi khuan. Ngoai ra, sd lugng va vj tri halogen hda cung cd anh hudng din dilm

boat tinh ciia permease enzymes can thiet dl hip thu cac hgp chit nay (Alexander and Aleem
1961;
Alexander 1999). Da cd nhirng bao cao cho thiy tinh dac hieu ca chit ciia nhirng vi khuin
68
nay ddi vdi cac loai thudc trir cd phenoxyalkanoic acid khdng bj khdng che bdi budc phan hiiy
enzim dau tien, vi vay, cd kha nang ban chat va vj tri ciia cac gdc thay the xac djnh sir hap thu
hgp chat vao trong te bao (Smejkal et al, 2001). Nhirng nghien cim ciia Leveau va cs. (1998) cho
thay protein phu trg chinh (MFS-major facilitator protein) tham gia vao sir hip thu dac hieu ciia
2,4-0 ciia chung Ralstonia eutropha JMP134. Nhirng vi khuan khac cung cd the cd protein van
chuyen ca chat dac hieu.
Aso Yuji va cs (2006) da thilt ke chimg Sphingomonas wittchn RWl tai td hgp, lam tang
kha nang phan buy dibenzofuran cua chimg gdc. Chiing tai td hgp dugc tilp nhan plasmid
pBEll mang cac gen aly, ccpA, algS, algMl, algM2, algQI, algQ2 va al-IV, va dugc nudi
trong he 2 pha Idng, 4 ngay trong mdi truong W cd bd sung dibenzofuran ndng do 10 mM.
Lugng dibenzofuran dugc them vao da hoan toan bien mat. Chiing RWl cung dugc nudi trong
mdi trudng cd dibenzofuran vdi ndng do 100 mM va lugng dibenzofuran nay cung mat hoan
toan sau 7 ngay nudi cay. Trong khi do chiing RWl dai phat trien kem ban va loai dibenzofuran
cham ban so vdi chiing tai td hgp. Chiing RWl(pBEll) da dugc sir dung de loai bd
dibenzofuran trong mdi trudng mdt each hieu qua.
Harcourt RL va cs (2002) da phat trien phuang phap dan gian de phan lap vi khuan phan
buy coumaphos. Ruifu Zhang va cs (2005, 2006) da nghien ciru cum gen ma hda cho protein
phan hiiy thudc trir sau ngudn gdc photpho hiru ca va chi ra rang chiing cau thanh transposon dj
hda va gdp phan phan tan gen giira cac vi khuan ban dja trong viing bj d nhiem.
Cd the thay rang, viee sir dung vi sinh vat de xir li du lugng thudc BVTV la mdt phuang
phap kha thi, hieu qua va kinh te. Phuang phap nay khdng nhii'ng lam giam du lugng HCBVTV
tren rau qua, ma cdn khdng gay d nhiem mdi trudng.
Vdi nhtrng ket qua nhan dugc d tren cd the thay cac chiing P va S16 cd phd ca chat rdng
han so vdi cac chiing SI, PL Cac chiing tuyen chgn deu cd kha nang sir dung de tao che pham
xir li du lugng HCBVTV ngoai ddng rudng.
4.

KET LUAN
Tir cac mau dat cd tien su sir dung thude BVTV da phan lap va tuyen chgn dugc 4 chiing vi
khuan (S16, SI, PI va RI) cd kha nang lam giam Regent va Sherpa tir ban 30% den ban 80%;
3 chiing khac (K5, LT33 va NT64) lam giam Pegasus tir 58,3% den 68,3% trong mdi trudng so
vdi ddi chirng sau 3 ngay nudi cay. Nhu vay cac chiing vi khuan tuyen chgn hoan toan cd tiem
nang sir dung trong xiir li du lugng Regent, Sherpa va Pegasus ngoai thuc dja.
TAI LIEU THAM KHAO
1.
Alexander M. - Biodegradation and Bioremediation, 2nd edition, Academic Press, NY,
USA, 1999.
2.
Alexander M. and Aleem M. I. H. - Effect of chemical structure on microbial
decomposition of aromatic herbicides. Agricultural and Food Chemistry 9 (1961) 44±47.
3.
Nguyin Thj Kim Ciic, Pham Viet Cudng, Nguyen Thj Tuyet Mai - Mdt sd tinh chat ciia vi
khuan phan buy Methyl Parathion phan lap tir cac mau dat tai Ha Ndi, Bao cao Khoa hgc
hgi nghj Sinh hgc Qudc gia :" Nhirng van de nghien ciru co ban trong Sinh hgc, NXB Dai
hgc Qu6c gia Ha Ndi, 8/2000, 29-34.
69
4.
Yuji Aso, Yukiko Miyamoto, Karen Mine Harada, Keiko Momma, Shigeyuki Kawai,
Wataru Hashimoto, Bunzo Mikami & Kousaku Murata - Engineered membrane
superchannel improves bioremediation potential of dioxin- degrading bacteria. Nature
Biotechnology 24 (2) (2006) 188-189.
5.
Harcourt R. L., I. Home, T. D. Sutherland. B. D. Hammockl, R. J. Russell and J. G.
Oakeshott - Development of a simple and sensitive fluorimetric method for isolation of
coumaphos-hydrolysing bacteria. Letters in Applied Microbiology 34 (2002) 263-268
6. Home I., Sutherland T. D Harcourt R. L., Russell R. J. and Oakeshott J. G. -
Identification of an opd (organophosphate degradation) gene in an Agrobacterium isolate,

Applied and Environmental Microbiology 68 (2002) 3371-3376.
7.
Leveau J. H. J., Zehnder A. J. B. and Van der Meer J. R. - The tfdK gene product
facilitates uptake of 2,4-dichlorophenoxyacetate by Ralstonia eutropha JMP134 (pJP4),
Journal of Bacteriology 180 (1998) 2237-2243.
8. Serdar C. M. and Gibson D. T. - Enzymatic hydrolysis of organophosphates: cloning and
expression of a parathion hydrolase gene from Pseudomonas dimimda, Bio/Technology 3
(1985)567-571.
9. Sethunathan N. and Yoshida T. - A Flavobacterium sp. that degrades diazinon and
parathion, Canadian Journal of Microbiology 19 (1973) 873-875.
10.
Smejkal C. W T. Vallaeys S. K. Burton
1,
and H. M. Lappin-Scott - A rapid method to
screen degradation abilirv in chlorophenoxyalkanoic acid herbicide-degrading bacteria.
Letters in Applied Microbiology 32 (2001) 273-277
11.
Ruifu Zhang; Zhongli Cui; Jiandong Jiang; Jian He, Xiangyam Gu, and Shunpeng Li -
Diversity of organophosphorus pesticide-degrading bacteria in a polluted soil and
conservation of their organophosphorus h\drolase genes, Canadian Journal of
Microbiology 51 (4) (2005) 337-343.
12.
Ruifu Zhang. Zhongli Cui, Xiaozhou Zhang, Jiandong Jiang, Ji-Dong Gu, and Shunpeng
Li - Cloning of the organophosphorus pesticide hydrolase gene clusters of seven
degradalive bacteria isolated from a methyl parathion contaminated site and evidence of
their horizontal gene transfer, Biodegradation, DOI 10.1007/s 10532-005-9018-6.
SUMMARY
ASSESSMENT OF REGENT, SHERPA AND PEGASUS DEGRADING CAPACITY OF
SOME ISOLATED BACTERIA
The use of pesticides has become an integral part of modem agricultural techniques, but has

also been developed for domestic and governmental purposes. Increased use of these chemicals
might have caused (or is strongly suspected to create) environmental pollution with impact on
ecosystems or, possibly, human health.
Pesticides are used widely in Vietnam with increasing quantity. According to reports of
Plant Protection Institute, the use and abuse of pesticides with high toxicity and even prohobited
ones in Vietnam lead to high residues of
those
in fruids and vegetables.
In nature, microorganisms are regarded as active factor in transformation cycle of chemical
elements and balance environment. Adaptation of indigenous soil microbial populations occurs
as a result of contact with xenobiotics. Many bacterial strains can degrade human made
70
xenobiotics belonging to the genera Flavobacterium, Pseudomonas, Agrobacterium, Bacillus.
Detoxification of pesticides by bacterial enzymes became focus of many investigations owing to
it's efficacy and economization.
The goal of this study was isolation and screening bacteria capable of decreasing Sherpa,
Pegasus, Regent concentrations in order to develop a bacterial formula for treatment pesticide's
residues in vegetables.
Four bacterial strains have been selected for treatment of Sherpa and Regent and 3 strains
for Pegasus. Strain RI had the highest activity against Sherpa, it eliminated 83.39% Sherpa in
culture medium after 3 days. Strain SI6 reduced Regent up to 76.76% comparing with control
after 3 days culture in minimal medium supplemented with 0.5 mg/ml Regent. Under the
conditions used, strain NT64 removed 68,38% Pegasus in culture medium.
The received results showed that RI and SI6 had wider substrate spectrum than SI and PL
Dia chi: Nhdn bdi ngdy 5 thdng 1 ndm 2008
Vien Cdng nghe sinh hgc, Vien KHCN Viet Nam.
71