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

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

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

TAP CHi KHOA HOC V A C O N G NGHE Tgp 47, s6 1,2009 Tr. 65-71 DANH GIA KHA NANG LAM GIAM DU LTONG SHERPA, REGENT VA PEGASUS CUA MOT SO C H O N G VI KHUAN TUYEN CHON NGUYEN THI KIM CUC, LE TH! HCNG MINH, PHAM VIET CUCNG 1. M O 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 C bd sung Pegasus, Sherpa va Regent dugc kiem tra kha nang sinh trudng ddng thdi tren moi O 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 Ten Regent Pegasus Sherpa SoTT Ten Regent Pegasus Sherpa chiing chiing 1 PI ++ ++ -I-+ 13 RIO ++ ++ + 0 P2 +-I- ++ ++ 14 Rll ++ ++ -i-i- 3 P ++ +-I- -I-+ 15 SI ++ ++ -I-+ 4 RI -H- -H- ++ 16 S2 + + +-I- 5 R2 ++ -l-l- + 17 S3 + ++ ++ 6 R3 ++ ++ -H- 18 S4 ++ -1- ++ 7 R4 -H- ++ ++ 19 S5 ++ + -I-+ 8 R5 ++ ++ + 20 S6 -1- ++ -I-+ 9 R6 ++ +-I- +-I- 21 S9 ± ++ ++ 10 R7 ++ +-I- -1- 22 SIO ++ + ++ 11 R8 -I-+ ++ ++ 23 S12 + ++ ++ 12 R9 -1-4- -l~l- ++ 24 S16 -H- -H- ++ 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 Ten chiing Ten du lugng Ket qua (mg/l) Phuang phap % giam so vdi DC 1 Diafcnthiuron 846,21 LC/DAD-FL 11,88 S16 (Pegasus) 679,17 29,27 SI 623,49 35,07 PI 882,58 8,09 RI 960,27 DC 2 S16 Cypermethrin 80,13 GC/ECD 75,50 SI (Sherpa) 146,82 55,10 PI 133,60 59,14 RI 54,31 83,39 DC 327,00 3 S16 Fipronil 21,82 GC/ECD 76,76 SI (Regent) 37,70 59,84 PI 64,03 31,80 RI 28,45 69,70 DC 93,88 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 Ham luang % giam so vdi Kha nang sinh trudng (mg/l) DC Pegasus Regent Sherpa K5 362.10 62,03 ++ ++ - NT64 301,52 68,38 -H- + - LT33 397,48 58,32 ++ ++ - DC 953,62 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