Tgp chiKhoa hgc TrudngDgi hgc Cdn Tho Phdn A: Khoa hgc Tu nhien, Cdng nghi va Moi trudng' 34 (2014)- 92-99
Tap chl Khoa hoc TrUdng €)ai hoc Can Thtf website: sj.ctu.edu.vn
PHAN LAP VA DINH DANH MOT SO DONG VI KHUAN BAN DIA PHAN HUY CHUYEN BIET HOAT CHAT PROPOXUR
Ttr NEN DAT BAO QUAN HANH TIM TAI THI XA VINH CHAU, TINH SOC TRANG
Do Hoang Sang', D6 Thi Xuan', Duong Minh Viln', Vo Thi Guang' va Nguyin Khoi NghTa*
' Khoa Nong nghiip & Sinh hgc U'ng dung, Trudng Dgi hgc Cdn Tha
Thong tin chung:
Ngdy nhdn: 17/06/2014 Ngdy chdp nhgn: 30/10/2014
Title:
Isolation and identification of specifically Propoxur degrading bacteria from soil samples in onion storage sites at the Vinh Chau town, Soc Trang Province
Tirkhda:
Propoxur, phdn huy, Paracoccus sp., vi khudn, VTnh Chdu
Keywords.\^^
Propoxur, degradation, Paracoccus sp, bacteria, Vinh Chau
ABSTRACT
Three soil samples from onion fields which had a history of intensive application of Propoxur at Vinh Chau, Soc Trang were collected for isolating bacteria being capable to degrade specifically Propoxur. Soil bacteria were enriched in mineral salt medium solution containing 30 ppm Propoxur as the only carbon source for bacterial growth. The whole procedure of isolation was established under the laboratory conditions on the shaker in dark with a total of 5 repeated generations. The results showed that two microbial communities coded as Pl-2 and P2-3 degraded well Propoxur (90% of the initial Propoxur concentration was degraded after 10 experimental days). Seventy-eight bacterial strains were isolated in total from these 2 potentially applicable microbial communities. Two out of four
selected strains which were coded as P23-7 and P23-26 degraded 100%
initial Propoxur concentration in the liquid solution after 4 incubation days.
According to the sequencing of gene 16S rRNA. these 2 Propoxur degrading bacterial strains were identified as a species specy of Paracoccus sp. P23-7 and Paracoccus sp. P23-26, respectively.
..-TOM TAT
Ba mdu ddt co lich sir sic dung hogt chdt Propoxur trong canh tdc vd bdo qudn hdnh tim lau ndm tgi khu vuc trdng hdnh dm tgi VTnh Chdu, Soc Trdng dugc thu thdp di phdn lap mgt sd ddng vi khudn co khd ndng phdn huy chuyen biit Propoxur. Vi khudn dugc ldm giau mat so tron^ moi truong khodng tdi thiiu long co bS sung 30 ppm Propoxur nhu la n^uon carbon duy nhdt Todn bg tiin trinh phdn lap dugc thuc hiin trong dieu kien phong thi nghiim, tren mdy Idc vd Irong tSi v&i long sd 5 ldn cdy chuyin lien luc. Kit qud nghien ciru cho thdy hai he vi sinh vgt ky hieu Pl-2 vdP2-3 thi hiin khd ndngphdn huy cao Propoxur (90% Propoxur sau 10 ngdy nuoi cdy). Tong cgng CO 78 dong vi khudn dugc phdn Igp tie hai hi vi sinh vgt ndy. Hai trong s6 bdn dong vi khudn dugc chgn di ddnh gid khd ndng phdn hiiy Propoxur trong moi trucmg khodng toi thiiu long co bo sung 45 ppm trong 18 ngdy ky hiiu P23-7 vd P23-6 phdn hdy 100% Propxur sau 4 ngdy thi nghiim. Dua vdo trinh tu gen 16S rRNA gidi ma cho thdy hai dong vi khudn ndy thugc ldp Prokaryote, Bacteria, Paracoccus vd dugc dinh danh ldn lugt nhu lodi Paracoccus sp. P23-7 vd Paracoccus sp. P23-26.
Tgp chi Khoa hgc Trudng Dgi hgc Can Tha Phdn A: Khoa hoc Tu nhiin. Cdng nghi va Moi irudng: 34 (2014): 92-99 I GIOI THIEU
Thi xa Viiih Chau tinh Soc Trang dupe biit din voi nghi ttflng hdnh tim va eu hdnh tim cung cap khflng chi cho Dflng bing song Ciiu Long ma con cho ci nuoc. Sin lupng hang nim khoing 20.749 tin nim 1994; dat 83.603 tin Uong nam 2004 v i khoang 130.000 tin vdo nim 2012. Hiennay,nang suit binh qudn cfta cu hinh tun li 14-15 tin/ha.
Difn tich ttong hanh tim duoc qui hogch giai doan 2016 - 2020 la 7.500 ha (Duong Vmh Hao, 2013).
Trong qud trinh canb tdc v i bdo quin h ^ giong nguoi ddn thuong sft dyng nhieu loai touoc bao ve toye vdt voi liiu cao dl phong tru siu bfnh Uong dfl CO boat chdt Propoxur (tin thuong mgi li Mipcin). Nong din tgi khu vye nay touong bao qudn hdnh giong sau thu hogch bdng cich tton hon hpp bpt Tale (Mg3(SiOio)(OH)2) vi Mipcin hoic Sherpa voi liiu lupng tmng binh 60 kg T^c + 3-4 kg Mipcin hogc 300-400 cc Sherpa dk xft ly (Nguyin Dftc Thing, 1999).
Hogt chit Propoxur dpc cho eon ngucri vi dpng vgt, hoa tan mgnh trong nude, khd nang hip phy ttln keo dat Id yeu va khd nang phin buy cua thuoc trong dit bdi vi sinh vat trong ddt cham (25 % Uong v6ng 100 ngdy Uong dit cdt). Propoxur lam bit hogt cdc enzyme acetylcholinesterase v i ldm te lift he than kinli cua con ttxing (Hayes, 1982; Berg, 1986). Cae toi nghifm nghiin eftu Uen dpng vat cho toiy Propoxur giy gidm sfl lupng con va Upng lupng thai Uin eho, mot so co hifn tupng quii toai, gay ra khfli u 6 bang quang vi tft cui^ ttln chupt khi nhiem man tinh 6 Iilu cao (NCEA, 1999).
Trong ddt, Propoxur e6 toi bi phin buy boi mot s6 vi sinh vgt nhu: Arthrobacler sp. (Nkedi-Kizza va ctv., 1992), Pseudomonas sp. (KarmanavaJli v i Bang 1: Mpt sS die dilm mo ta cua 3 mau dat thi nghiem
Nmnerkar 2000), Streptomyces spp. (Rahmansyah vd ctv., 2012). Mgc dft, vifc ftng dyng cong nghe vi suto ttong xft ly dflc chit 6 nhiem moi ttorong ddc bilt Uoi^ xft ly dit 6 nhiem touflc bio vf thyc vgt dupe ftng dyng rflng rii Uen toi gioi, nhung a Vift Nam vin dk nghiin cftu niy cdn ban chi. 6 DBSCL mpt s6 c6ng trinh nghiin eftu vi ITnh vyc nghien cftu niy di dupe cflng bfl gdm: nghien cftu vi phdn Igp vi khudn phan buy hogt chat trft co 2,4- D (Nguyin Thi Phi Oanh vd ctv., 2011), hogt chit toudc trft sau Chlorpyriphos etoyl (Nguyin Thj Lan Hucmg, 2012), hoat chit touoc trft cd Pretilaehlor (Nguyin Thi Td Quyen, 2013) vi boat chit kieh thich ra hoa ciy an trai Paelobuttazol (Ding Pham Thu Thio vd ctv., 2014). Tuy nhiin, chua cd nghien cftu nio vi phdn l§p va tuyin chpn mpt so ddng vi khuin cd khi ning phdn buy hogt chdt Propoxur. Do dd, nghiin cftu nay dupe toye hifn nhdm myc tieu: Phdn l|p, toyen ebon vi djnh danh mpt s6 ddng vi khuin bdn dja cd khi ning phdn hfty hogt chdt Propoxur tft nen dit bdo qudn hdnh tim tai thi xa VTnh Chiu, tinli Sdc Trdng.
2 PHU'OfNG PHAP NGHIEN Ciru 2.1 Ngu6n vi khuan
Ba mdu dit Ic^ hieu Pl-2, P2-3 vd VH-4 dupe thu toap hr nin ddt canh tic vd klio bdo quin hinh tim tgi Phudng 1, Phudng 2, xi Vuto Hii ciia thj xd VTnh Chiu, tmh Sdc Trang dftng dk phin Igp mOt sfl ddng vi khuin cd khd ning phin buy chuyin bilt Propoxur vi nflng ddn tgi ba diim liy mlu ndy sft dung hogt chit Propoxur mSi ndm di tring vd bao quin hinh tfm. Miu dit dupe liy voi dp sdu 0- 20 cm. MOt sd todng tin vi 3 dia diim thu mdu dit duoc ttinh bdy trong Bing 1.
MSU
dit Nin dit Dia diem Tbdi gian sur dung
Propoxur (nam) P12 Bio quin hdnh tim gidng Phudng 1,
P23 Bdo quin hinh tim giflng Phudng 2, VH4 Canh tie hanh tim VTnh Hai,
Vinh Chdu, Sdc Trang 40 VTito Chiu, Soc Trang Liu nim VTnh Chdu, Sdc Trang 3 0 2.2 Phan Igp cac ddng vi khuan ban dia co
tiim nang phan hiiy hogt chat Propoxur tu 3 mau dat thu thgp
M^t so vi sinh vdt ban dau ttong cae mdu dit dupe xdc dinh theo phuong phap hda loang eua Ian va Charles (2004). Sau khi da ttii dich vi khuin lln bl m|t, cic dTa mdi Urrdng nufli ciy duoc dat vio ttii nylon vi ft d nhiet dp phdng. Sd khuan lgc dim dupe ttln bl mat dia sau 3 ngay u se dupe quy doi
toeo hf s6 pha loang v i toe tich di xic dinh m^t sd vi khudn ban dau cua cae mdu dat.
Cin 1 g dit (ttpng lupng khd kift) cho vio binh tam gidc tilt trftng chfta 24 mL dung dich khodng toi toiiu cd bfl sung 30 ppm Propoxur. Thdnh phan cfta mdi ttudng khoang tdi toilu trong 1 L dung dich nhu sau: 3,75 g K2HP04, 1 g KHjPO^, 0,25 g NaCl, 0,1 g MgS04.7H20 vd 0,01 g CaCb-HjO.
Moi trudng dupe khft tning d I 2 r c , 20 phiit trong
TQP chi Khoa hgc Trudng Dgi hgc Cdn Tha Phdn A: Khoa hgc Tv nhien, Cdng nghi vd Mdi Irudng: 34 (20N)- 92-99 noi hdp tilt trung sau dd bd sung 10 mL dung dich
VI lirpng. Thanh phdn vi lupng ttong 1 L dung dich nhu sau: 10 mg Na2Mo04.H20, 25 mg H2BO3, 15 mg ZnClj, 5 mg CuCb, 10 mg FeCU. Cac miu nuoi cay dupe dat Uen may lie vdi tdc dfl 90 rpm (nhim tgo kha nang uao ddi oxy tdt eho dung djch) d dilu kiln nhift dd phdng toi nghiem va ttong tdi.
Ddy dupe xem li toi hf nudi cdy diu tiln. Sau 7- 10 ngiy nudi ciy, hut 1 mL dung dich nudi cdy va lpc qua mdng lpc tilt trung, sau dd, xac dinh nflng dp Propoxur ttong miu tt^n he toflng HPLC (Sbimazu-LC20A) vdi cdc todng sfl nhu sau: sft dyng cpt C18, tf If pha dpng aeetonitrile : nude ttrong ftng la 45:55, budc sdng 214 nm, luu lupng eua pha d^ng 1 ml/phut, todi gian xac dinh pho Propoxur li phftt thft 7,5. Vifc nufli va ehuyin sang toe he mdi chi toye hiln khi ndng dp Propoxur ttong miu giam ban 50%. Toan bp qui trinh nufli cdy dupe l§p lgi ttong 5 lin lien bac. Sau lin nudi cdy toft 5, 100 n-L mdi ttudng nudi ciy vi khuin vdi cac ndng dO pha lodng khic nhau dupe trdi lln trin be mit mfli trudng agar TSB (gom 30g Tryptone soya broto v i 15g agar cho 1 L nude cit) di phin l^p cdc ddng touin v i tiin hdnh khio sit cie die tinh ve hinh toai khuin lgc vd hinh dgng ti bdo. Gram vi dgc tinh oxidase.
2.3 Dinh gii Idia nang phin huy hogt chat Propoxur eiia 2 hf vi sinh vit 1^ hieu Pl-2 va P2-3 trong mfli trudng khoing tdi thilu long
Trong qui ttinh phan Igp d muc 2.2 qua quan sat cho toiy hf vi sinh viit 1^ hieu VH-4 da khdng the hiln kha ning phdn huy tflt boat chit Propoxur nen he vi sinh vgt nay da dupe logi ra vd chi edn lai 2 hf vi sinh vgt ky hifu: Pl-2 vd P2-3 dupe sft dyng cho vifc phdn Igp cie ddng vi khuan ed tiera ning phin hfty Propoxur. Khi ning phin hfty Propoxur cfta 2 he nay dupe ddnh gia trudc khi tien hanh phdn lap ddng vi khuan tren dia agar. Thi nghifm dupe thyc hifn nbu sau: hftt 1 mL dich vi khuin d thi hf nudi cdy toft ndm cho vdo binh tam giac 100 mL cd chfta 24 mL dung dieb khoang tdi thiiu bd sung 30 ppm Propoxur. Thi nghifm dupe tiin hdnh vdi 3 ldn lap lgi cho moi hf vi sinh vdt vi n ^ i l m thfte doi chung dupe tien hanh tuong ty nhung khdng chung hf vi sinh vgt. Cac mau dupe lie Uin mdy lie vdi toe dp 90 rpm, d nhift dp phdng toi nghifm, ttong tdi vd ttong 10 ngiy.
Cic chi tilu toeo ddi Uong thdi gian bd tri thi nghiim bao gflm: 1) Nflng dp Propoxur vio cdc todi diim: 0, 1, 5 vi 10 ngdy sau bd Ui thi nghifm;
2) Mat s6 vi khuan ttong dung dich mdi trudng nudi ciy vio eae todi diem: 1, 5 vi 10 ngiy sau khi bd tri toi nghiim. Mat sfl vi khuin dupe xdc dinh bdng phuang phdp nhd gipt ttln mdi trudng TSB.
DTa chfta vi khuin dupe dgt vio tiii nylon vi ft ttong tu ft vdi nhiet dp 30°C ttong ba ngdy. Sau dd, dim mgt sd khuan lgc hien dien Uen bl mgt dia vi tt'nh mit sfl vi khuin vdo cic todi dilm tou miu.
2.4 Dinh gii va so sinh khi nang phin buy Propoxur cua 4 ddng vi khuan tuyen chpn trong moi trudng khoang toi thilu Idng
Bdn trong tdng sd 78 ddng vi khuin phdn ldp ky hifu: PI2-3; P23-7; P23-19 vi P23-26 duoc tuyin chpn dl tiin hanh danh gid kha ndng phdn buy Propoxur cfta chung. Bdn ddng vi khudn ndy dupe idm giau mat s6 Uong mdi trudng GYE (glucose yeast extract) ttong 3 ngiy ttln may Idc.
Tbinh phdn ciia mfli trudng GYM ttong 1 L dung dich bao gdm: 10 g glucose va 10 g yeast extract Sau dd, sinh khfli vi khuin duoc thu hogch. Hieu chinh dp dye ciia dung dich chfta vi khuin vdi nude khft khodng tift trftng bang mdy do quang phd vi OD 600 nm = 0,7. Hut ImL dich vi khuin da hieu cbinh dp due cho vio binh tam giac 100 mL chfta 24 mL dung dich khoang tdi toilu cd bd sung 45 ppm Propoxur. Thi nghiim dupe tien hdnh vdi 4 l§p lai.. Cdc mau dupe lac trin miy 15c vdi tdc dp 90 rpm, d nhift df phong thi nghifm, Uong tdi vd ttong 18 ngay.
Cdc chi tieu theo ddi ttong thdi gian bd tti thi oghifra bao gdm: ndng dp Propoxur vi mat sfl ddng vi khuan vdo cdc thdi diem: 0; 1; 4 va 18 ngiy bo tii thi nghifm (tham khao myc 2.2 vi 2.3 cho phuang phdp xdc djnb ndng dp Propoxur Uong mdu nudi cay.^yixac.dinh m#t sfl-vi kbuan).
2.5 ' Giailna'trinh''*^gMrtSrRNA va xac djnh if muc dfl loai cua hai dong vi khuan ky hiiu P23-7 va P23-26
DNA cua 2 ddng vi khuin ky hifu P23-7 vi P23-26 thi hiln pban hfty cao boat chit Propoxur Uong mdi Uudng khoing toi toilu idng (myc 2.4) dupe Uich toeo quy Uinh cua Ihrmark vi ctv., 2012. Sau dd, sin phim trich DNA dupe khuech dgi bing phdn ftng PCR vdi vdi bd mdi 27F-907R (Xuan vd ctv., 2012) cd Uinh ty nhu sau:
27F 5' AGA GTT TGA TCC TGG CTC AG 3' 907R 5" CCG TCA ATT CCT TTR AGT TT 3' Hai doan moi ndy nhdm vao doan gene 16S
Tgp chi Khoa hgc Trudng Dgi hgc Cdn Tha Phdn A: Khoa hgc TV nhiSn, Cdng nghi vd Mdi Irudng: 34 (2014): 92-99 rRNA cua vi khuin. Chu ttinh nhift cho phin ung
PCR: giai doan so khdi 94°C (3 phut), 25 chu ki:
92°C (30s)-56x (45s)-72°C (30s), 7 2 ^ (7 phut);
tru sdn phdm d 4°C (Xuan vd ctv., 2012). Sdn phdm PCR dupe kiim tra Uin gel ^arose 1,5%
trudc khi giii Uinh ty,
Cic hda chit di thuc hifn phan ftng PCR bao gflra (thi tich/1 phin ftng): 4pL Dream taq buffer (5x); 0,4 pL mfli xudi 27F (10 pM); 0,4 pL mfli ngupc 907R (10 pM); 6 - 10 yL DNA tinh sach;
2,5 pL mQ-HzO, 0,4 pL dNTP (lOmM); 2,2 nL MgCh (25 raM); 0,1 pL Dream taq (5 U/pL). Kit qud gidi trinh ty dupe so sinh vd dd tim tten ngin hdng gene thi gidi tten trang web http://blast.ncbl.nlm.nih.gov/Blast.cgi dk xic dinh mftc dp lodi cua hai ddng vi khuin khio sdt.
3 KET QUA VA THAO LU^J*
3.1 Phan Igp cic ddng vi khuin cd tilm nang phan buy Propoxur tir 2 he vi sinh vit thi hien khi ning phin hiiy tot Propoxur
Kit qud mat so vl khuin ban ddu cfta 3 mdu dat thi nghiim dupe ttinh biy Uong Bing 2. Cd sy khac nhau vi mit sfl vi khudn ban diu cfta 3 raiu dit thu toip. Mat sd vi kbuan cao nhit d miu dat Pl-2, ki din Id P2-3 vd toip nhdt d miu VH-4.
Bang 2: Mat Mlu dSt Pl-2 P2-3 VH-4
so ban dan cua 3 man dat thi nghidm CFU.r' 2,1x10' 2,2x10' 9,5x10' Sau tooi gian ldm giiu m$t sfl va phan lap vi khudn cd tiim ning phdn huy Propoxur ttong mdi trudng Idng va agar tft 2 hi vi sinh vdt k j hiiu Pl-2 vi P2-3, tdng c^ng 78 ddng vi khuin di dupe phin l$.p. Trong tdng s6 78 ddng vi khudn dupe phin liip cd 38 ddng tft hfvi sinh vgt ki hieu P12va 40 ddng tft hf vi sinh v§t ki hifu P23. Tft 78 ddng vi khu^
phin lap, dupe nhdm lgi thdnh 15 nhdm dya Uen die diim hinh toii khuin lac. Hinh dgng te bao cfta 15 ddng vi khuin dgi diln cho 78 ddng phin l$p dupe quan sit dudi kinh hiin vi vi kit qua ghi nhdn cd 3 dgng hinh nbu sau: que ngan, que dai vi hinh ciu (Hinh 1). Nhdm I: dang ti bdo hinh que ngin gflm cd 8 ddng k^ hifu: P12-7, P12-17, P12- 27, P12-37, P23-7, P23-20, P23-26 v i P23-29 (8/15 ddng, chiim 53%). Nhdm 2: dgng tl bao hmh que dai gdm 2 ddng ky hieu: P12-24b' va P23-18 (2/15 ddng, chiim 13%) va Nhora 3: dgng ti bao hinh ciu gdm cd 5 ddng ky hifu: P12-3, P12-34, P23-25, P23-27 vd P23-35 (5/15 ddng, chiim 34%).
(A) (B) (C) Hinh 1: Ba hinh dgng te bao vi khuan dien hinh dupe quan sit trin kinh hien vi (A): Hinh cau;
(B): Hinh que ngin va (C): Hinh que dii (quan sit d ElOO) Kit qud kiem tra ve gram vi khudn cho toay
nhu sau: 9 ttln tflng s6 15 ddng vi khuan dupe phin loai nhu la vi khuin gram duong (60%), cdn lai 6/15 ddng vi khuin dupe phdn logi nhu la gram am (40%). Cuoi cftng, kilm Ua khi nang oxidase cua vi khuein de xdc djnb vi khuin cd enzyme cytochrome oxidase hay khdng. Vi khuan toi hifn phin ftng duong tinh (positive) khi test oxidase cd thi sft dyng oxi tham gia vdo chudi dan truyen difn tft, sinh ATP ttong qui trinh hd hip, kit qui kiim Ua khd ndng oxidase cua 15 ddng vi khuan dgi diln eho toiy nhu sau: 8/15 ddng vi khuin thi hiln duong tinh vdi phin ftng oxidase vi cdn lgi 7/15
ddng vi khudn the bifm am tinh vdi phin ftng oxidase.
3.2 Dinh gii kha nang phin hfty Propoxur trong dung djch khoing toi thilu cua 2 hf vi sinh vgt kf hifu: Pl-2 vi P2-3
Kit qua vi khd ning phan buy Propoxur cfta 2 he vi sinh vit ky hlfu Pl-2 v i P2-3 Uong mdi trudng khodng tdi thiiu Idng dupe ttinh bay trong Hinh 2. Nhin chung, ning df Propoxur d cdc nghifm toftc, bao gdm cd nghiem toftc d6i chftng gidm din toeo todi gian nudi cdy. Khi nai^ phin buy Propoxur cua cd 2 he vi sinh vat khdc bilt
Top chi Khoa hoc TrudngDgi hgc Cdn Tho Phdn A: Khoa hgc Tu nhiin, Cdng nghi vd Mdi Irudng: 34 (2014): 92-99 khdng cd y nghTa todng kl (p>0,OS) tgi cic tocri
diem thu mau. Tuy nhien, ndng dp Propoxur Uong cd 2 hf vi smh vit thip hon v i khdc bilt ed y nghia thdng ke so vdi nghiem toftc dfli chftng (p<O.OS).
Nong dp Propoxur giam nhanh vao todi diim 5 ngay sau khi nudi ciy, tu 30 ppm xuflng cdn 7 ppm 6 cd 2 he vi sinh vgt. Sau 10 ngdy nudi cdy, ndng
dp Propoxur d cd 2 he vi smh vit gidm xudng dudi 5 ppm. Su gidm ndng dp Propoxur ttong nghiim touc ddi chftng dupe giai thich li do Propoxur cd toi bi touy phin (1,5%/ngiy) Uong mfli trudng nuac cd pH trung tmh (pH=7.0) (Hartley vi Kidd, 1983).
Thoi stall uuoi cay (usay)
Hinh 2: Kha nang phan huy hogt chat Propoxur ciia 2 he vi sinh vit ky hieu: Pl-2 va P2-3 trong dung dich khoang toi thieu bo sung 30 ppm Propoxur sau 10 ngay nudi cay (n=3 v i sai so chuin)
Kit qua the hifn sy phat ttiin mat so vi khuan theo thdi gian nudi cdy dupe Uinh biy trong Hinh 3. Nhin chung, mat sd vi khudn e6 xu hudng ting din toeo todi gian nufli cdy. Giai dogn tft 1 din 5 ngiy mat sd vi khuin ting nhanh nhat, sau do dn dinh vi sau. Khdng cd sy khac bift toing kl vl mat sd vi khuin gifta 2 hf vi sinli vit ki hieu Pl-2 vi P2-3 d tit c i cdc tool diem thu mdu. Sy khflng khac bift vl mat sd vi khuan gifta 2 hf vi sinh vat vio cae todi liy mdu la co sd phft hpp di gidi thich cho hiln tuong khflng khac bift vi khd ndng phin
huy Propoxur eua 2 hi vi sinh vgt vao cic todi diim thu raiu. Giai dogn 1-5 ngdy sau khi bfl tri toi nghifm rait sd vi khuan ting lln rit mgnh, ddng thdi, ndng dp Propoxur trong moi tnrdng nudi cdy cung giim rapt lupng rdt ding kl, dilu ndy chftng td, vi sinh vdt da sft dung ngudn carbon tft Propoxur cho smh trudng v i phit triln mat sd. O nghifm thfte ddi chftng khdi^ cd bat eft vi khuan nio tira tody Uen dia agar, dieu niy cho toay cae toao tac ve tift Uiing Uong qua trinh bfl Ui thi nghifm di dupe bio dim vi ddng tin cdy.
P2.' Pii'l'iiMll -•" l>|il1l
ThM elan nnof o y (nciy)
Hinh 3: Sy phat trien mgt s6 vi khuin trong dung dich khoang tdi thieu bo sung 30 ppm Propoxur trong 10 ngay nuoi cay (n=3 va sai so chuan)
Tgp chi Khoa hgc Trudng Dgi hgc Cdn Tho Phdn A: Khoa hgc Tv nhiin, Cowg nghi vd Mdi Irudng: 34 (2014): 92-99 3.3 Danh gia kha ning phan buy Propoxur
trong moi trudng khoing tdi thieu Idng cua 4 ddng vi khuan dur^c tuyln chpn
Kit qui ddnh gia khi ning phdn buy Propoxur Uong mdi trudng long sau 18 ngdy toi nghiem cua 4 ddng vi khuin ky hieu: P12-3, P23-7, P23-19 va P23-26 dupe ttinh bdy d Hlnb 4. Kit qua eho thdy ttong 4 ddng vi khuin thft nghifm toi chi cd 2 ddng ky hifu: P23-7 vd P23-26 la thi hiln khi nang phin buy rit tflt Propoxur Uong dung dich khoang tdi thiiu khi so sinh vdi nghifm touc ddi chftng,
I
ttong khi dd cic ddng vi khuan edn 1^ kbi nang phdn buy Propoxur kem hon b ^ ddng ndy va phan buy Propoxur khdng ding ki so vdi nghiem toftc ddi chftng. Ddng vi khudn 1^ bifu P23-7 phan buy dupe 30 ppm Propoxur ttong mdi trudng long sau 1 ngdy thi nghifm vd sau 4 ngay nudi cay ndi^ dfl Propoxur trong mfli trudng d i bi phdn hiiy hoin todn. Trong khi dd, ddng vi khudn ky hieu P23-26 chi phin buy 10 ppm Propoxur sau 1 ngay nufli cay. Tuy nhien, vdo ngiy thft 4 ddng vi khuan ndy da phin buy hoin todn Propoxur.
51
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Hinh 4: Kha nang phan hiiy Propoxur cua 4 dong vi khuin tuyen chpn trong dung djch khoang toi thieu bo sung 45 ppm Propoxur sau 18 ngay nuoi cay (n=4 vi sai s6 chuin) Ket qui vi sy phit triin mit so cfta 4 ddng vi
khuin thft nghiim ttong mdi ttudng nudi cdy khoang tdi thieu Idng bo sung 45 ppm Propoxur Uong 18 ngay dupe ttinh biy ttong Hinh 5. Kit qua eho thiy nhu sau: Mgt sd cua tit ca cac ddng vi khudn thft nghifm deu cd xu hudng tang toeo todi gian. Mit sfl vi khuin ddng P23-26 d ngiy toft nhdt tiiip hon so vdi ddng P23-7. Dflng todi, khi nang phdn huy thuoc Propoxur cua ddng P23-26 cung toip ban rit nhiiu so vdi ddng P23-7 vao ngay 1.
Tuy nbiln, d ngiy toft 4, mat sd vi khuan ddng
P23-26 tang len mgnh va khflng khdc biet thdng kl so vdi ra|it s6 ddng P23-7. Bin cgnh dd, kit qud phin tich nflng df Propoxur ttong mdi trudng Idng cfta 2 ddng vi khudn nay deu bang 0 ppra vdo ngdy toft 4. Diiu nay rd rdng cho thiy, sy gia tang radt sd cua 2 done ndy Id nguyin nhdn ddn din sy g i ^ ndng dp touoc Propoxur Uong mdi trudng nufli ciy v i 2 ddng vi khuin niy di su dung Propoxur nhu la ngudn carbon cho viec tang mat sfl, sinh trudng va phat triln.
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Hinh 5: Sy phit triin mat sfl cua 4 ddng vi khuin thft nghifm trong dung dich khoing thi thilu bo sung 45 ppm Propoxur sau 18 ngay nuoi ciy (n=4 vi sai so chuin)
Tgp chi Khoa hgc Ihrdng Dgi hgc Cdn Tha Phdn A: Khoa hgc Tu nhien, Cdng nghi vd Moi Irudng: 34 (2014): 92-99 1°"^ '^'' ^^^ '^^^ ^ nghiem eho toiy 2 ddng vi
khudn phan lip uong nghiin cftu nay k^ bilu P23- / va P23-26 toi hifn kha ndng phdn huy chuyen bift Propoxur Uong mdi ttudng khodng tdi thiiu Idng va ea 2 ddng niy cd tilm nang ftng dung cao ttong vifc xft ly dit bj d nhilm vdi Propoxur va nln dupe chpn cho nhftng nghiin cuu tiip toeo.
3.4 Giai mi trinh t y g e n i e S rRNA va xac djnh d mftc dp loii ciia hai ddng vi khuin ky hifu P23-7 v i P23-26
16S rRNA vdi co sd du lifu ttln ngin hdng gene thi ^di. Kit qui cho toiy trinh ty dogn gen ciia vi khuin P23-7 vi P23-26 Iin lupt tuong ddng vdi doan gene 16S rRNA eiia loai Paracoccus sp. dong Y3B-I (sd dai^ ki: HM018693.1) vi Paracoccus sp. SMIC-4 (sd dang ki: FJ877155.1) vdi dd tuong ddng lin lupt la 100% va 99%. Nhu vgy, 2 ddng yi khuin ky hifu: P23-7 v i P23-26 dupe sdp xip toeo bac phin loai nhu sau: Prokaryote, Bacteria Proteobaeteria, Alfrfiaproteobacteria, Rhodobaeterales, Rhodobacteraceae, Paracoccus, Paracoccus sp.
(Bang 3).
Hai ddng vi khudn ky hifu P23-7 vd P23-26 29 dupe giii ma trinh ty va so sdnh Uinh ty doan gene
Bang 3: Dgnh danh cic ddng vi khuin thi hien su phan hiiy Propoxur theo dp tuong ddng cua doan gen 16S rRNA
CTT r*A M * •= Dotuofng Cac done vi khuan trin Cff sddij'lifu T,. . j L STT Ddng Nguon goc ^i ,„,= 5-= 7 —-" Dmh danh
^ 6 6 aong (%) Vi khuan So dang ki Phudng 2,
VTnh Chdu, Sdc Tring
Paracoccus sp.
Y3B-1
Paracoccus sp. P23-7 Phudng 2,
VTnh Chdu, Sdc Trdng
Paracoccus sp.
SMlC-4 FJ877155.1 Paracoccus sp. P23-26 4 K E T L U I N
Hai mdu dat k^ hifu: Pl-2 vi P2-3 thu todp tft nin ddt ttong kho bio qudn hdnh tim tgi thj xd VTito Chdu, tinh Sdc Trdng cd chfta ngudn vi khuan cd khi ning phdn hfty rit cao va chuyen biet Propoxur. Hai dong vi khuin ky hieu P23-7 v i P23-26 toi hifn klii ning phan hfty rit huu hifu Propoxur trong dung dich khodng tii thiiu vd dupe xem nhu Id 2 ddng cd tiim ndng ung dyng eao nhit trong vile xft ly dit d nhilm vdi hogt chit Propoxur vd ca 2 dupe dinh danh ldn lupt g ^ n h u la lodi Paracoccus sp. P23-7 va Paracoccus
sp. P23-26.
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