Tgp chi KHOA HOC DHSP TPHCM Hoang Thi Hdng vh tgk
PHAN LAP VA TUYEN CHON CHUNG XA KHUAN Tir RlTNG NG^iP MAN CAN GICJ KHANG N A M FUSARIUM SP.
H O A N G TH! HONG*, NGUYEN NGOC PHL/aNG"
TOM T A T
Chung toi da tien hanh phdn lap duac 55 chung xa khudn khac nhau tir rimg ngap man Cdn Gid vd phdn thdnh 3 nhdm; nhom I: Trdng, gom 19 chiing (34,5%); nhom 2:
Xam - ndu - den, gom 25 chung (45,5%); nhom 3: Vdng nhgt - vdng - vdng ndu, gom II chung (20%). Tir bg suu tap tuyen chgn dugc chung xa khudn F46 co khd ndng Ididng ndm Fusarium sp. mgnh. Qua cdc nghien cuu dd xdc dinh duac cdc dieu hen nuoi cdy thich hap cho chung xg khudn ndy sinh chdt khdng ndm Fusarium sp. nhu sau: cao thit 4g, peptone 4g, cao ndm men Ig, maltose lOg, nuac cdt i lit; NaCl 1%; pH = 5-6; 25°C; 84 gid.
Tk khda: phdn ISp xa khuan, chat khang nam, Fusarium sp.
ABSTRACT
The isolation and selection of actinomycete species from Can Gio tropical swamp for their antifungal feature against Fusarium sp.
We have isolated 55 different species of actinomycetes from Can Gio tropical swamp and divided them into 3 groups: group I called White, including 19 species (34.5%), group 2 called Gray - Brown - Black, including 25 species (45.5%), group 3 called Light yellow - Yellow - Brownish yellow, including II species (20%). From this collection, we have succeeded in choosing an actinomycete called F46 whose antifungal feature is very strong.
We have found the best planting conditions for this species to produce antifiingal is as following: 4g. peptone 4g, yeast extract I g, maltose 10 g, distilled water 1 litre; NaCl I %;
pH = 5-6, 25°C; 84 hours culture
Keywords: isolation of actinomycetes, antifiingal, Fusarium sp.
1. Mo* dau
Theo thong ke cua to chiic Nong - Luong The gidi cho thay cac loai cay trong tren dong rupng hien nay phai chong dd vdi khoang 100.000 loai sau, 10.000 loai nam, 200 lo^i vi khuan, 600 loai tuyen trung va 600 loai virus gay hai khac nhau. Day qua la mOt l\rc lupng hung h^u tan cong cay trong, gay ton that dang ke cho miia mang, udc tinh lam mat trSng khoang 20% san lupng luong thuc thuc phim hing nam. [5]
" CN, Trudng Ogi hpc Su phgm TPHCM
" ThS, Trub-ng Dgi h<?c Su phgm TPHCM
Tap chi KHOA HOC DHSP TPHCM SS 51 nam 2013
Mgt trong sd tac nhan gay hai cay ttdng nguy hidm trong san xuit la nhdm nam cd ngudn gdc trong dit nhu: Fusarium, Phytophthora, Rhizoctonia, Pythium,... Nhdm nim nay cd phd ki chu rdng, gay hai tten nhidu loai cay trdng khac nhau nhu: cac cay thudc hg d?iu, biu bi, ngd,... Dac biet, la chi Fusarium gay benh heo vang lam ciy chet nhanh, mdt sd cdn san xuit ddc to gay nguy hidm cho ddng v?t va con ngudi. Nam Fusarium cd thd tdn tai nhidu nam ttong dit, ttong tan du thuc vat va lan truyen qua hat gidng, cay nhidm benh hoac lan ttniydn theo nudc va gid. Thdi tiet am ap, nhiet do tiimg binh 27°C - 30°C, do im ttong dit tuong ddi cao la didu kien thich hgp oho su phat tridn ciia nim gay benh [1]. Benh phat tridn nhidu tten ddng rudng tir thang 3 din thang 11 va gay hai manh nhit vao thang 9 den thang 11. [6]
Dd ban chd nhflng thiet hai do nim benh gSy ra, con ngudi da ap diing rat nhieu bidn phap trong cdng tac bao ve cay trdng. Trong do, co thd ndi khdng mdt bien phap bao vd miia mang nao hihi hi?u hon bien phap hda hoc vd mat hidu qua va quy md [5].
Tuy nhien, bien phap hda hge ciing nay sinh ban che cua nd nhu: hien tugng nhdn thuoc d cac tac nhan gay hai, gia tang d nhilm mdi truong (MT), mat can bang sinh thai, chat hda bgc tdn du trong dat gay nguy hiem cho siic khde con nguoi. De khac phuc nhung ban che cua bien phap hda hge ttong cdng tac bao ve cay trdng khdi nam gay hai, cac nha khoa hoc da khdng ngimg nghien ciiu va cho ra ddi che pham thudc trii nim cd ngudn gdc sinh hpc. [6]
Che pham trir nam cd ngudn goc sinh bgc cd nhieu uu viet hon so vdi thudc hda bgc, cu thd la: khdng gay h^ii cho con nguoi, vat nudi, cay ttdng; cho ddn nay chua phat hien dugc hien tugng nhdn thudc va nhanh chdng phan buy, khdng gay d nhilm MT [6]. Cd rat nhieu ddi tugng vi sinh vat (VSV) dugc quan tam de san xuat cac chd pham sinh hge, mdt ttong sd dd la xa khuan vi kha nang ddi khang cua chiing vdi VSV kiem dinh. Trong nhiing nam gin day, nhidu nha khoa hpc ttong nudc cflng nhu tren thd gidi dac biet liru y ddn viec tim kidm cac hgp chat sinh truang thii cip tit xa khuin rimg ngap man (RNM). Bdi theo cac nha khoa bgc, chiing la ngudn sinh cac chat cd hoat tinh sinh hge ddi dao, frong dd cd chit khang nim. Thuc td, hg da phat hi?n nhilu hgp chit khang khuan, khang nim, khang virus, khang ung thu,... tu xa khuin RNM vol boat tinh manh hon rat nhieu so vdi xa khuan tu dit liln.
2. V|t lieu va phvang phap nghien cihi 2.L V^llieu
De tai da sii dung cac gidng vi sinh vat nhu sau:
Xj khuin phan lap tu dit KNM huydn Cin Gid, Thanh phd Hd Chi Minh.
Vi sinh vat kiem djnh gdm: Nam Fusarium sp. nhan tii bd suu tap gidng ciia Vien Cdng nghd Sinh hoc Trudng Dai hpc Ndng Lam, Thanh phd Hd Chi Minh.
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Tap chi KHOA HOC DHSP TPHCM Hoang Thi Hdng va igk
De tai da sii dung cac MT: MT Gause I, MT Gause II, MT Emerson, MT PDA, MT ISP4.
2.2. Phuffngphdp nghiin cuu
Cac m^u d4t thu duoc bing phuong phap liy miu dugc bao quan a tu lanh A°C.
Tien hSnh phan lap xa khuan theo phuong phap phan lap xa khuan cua Vinogradski [3]
va bao quan trong ong thach nghieng chua 5 ml MT Gause I (MT dac), sau 6 ngay thu hoat tinh khang nam Fusarium sp. bang phuong phap cay cham diem ciia Crawford, 1993 [7] (X? khuan ducrc nu6i 3 ngay tren 1 canh ciia dia MT PDA, canh con lai ciy cham diem nam Fusarium sp. tir dia MT PDA khac nuoi trong 3 ngay, danh gia kha nang urc che nam dua vao khoang each vo khuan giiia nam va xa khuan). Tuyen chpn chiing CO hoat tinh manh nhat.
Chung xa khuan F46 dugc mo ta dac diem hinh thai bang phuong phap quan sat dfii thS va quan sat vi the (phuong phap xe ranh thach cua Okamia Suzuki, 1968).
Chung xa khuan F46 dugc nuoi trong cac binh tam giac 250 ml chiia 50 ml cac MT khac nhau (MT Gause I, MT Gause II, MT ISP4, MT Emerson dang long), thay doi cac dieu kien nuoi cay nhu nguon carbon, nguon nitrogen, pH, nong do NaCl, nhiet do, ih.b\ gian va thu hoat tinh khang nam theo phuong phap khoan 16 thach (Sii dung khoan mit chai vo triing, khoan 16 tren dia MT PDA. Diing que c^y v6 trung, cay cham diem nam Fusarium sp. dugc nu6i 3 ngay tren MT PDA (dac) vao 2 phia d6i dien ciia 16 th^ch, sau do hiit 0,1 ml dich nu6i cay xa khuan 6 ngay thu 3 nho vao 16 thach mdi khoan. De 6 nhiet do phong, sau 3 ngay tien hanh kiem tra ket qua dua vao khoang each vo khuan). [4]
3. Ket qua va bifn lu^n
3.1. Ph&n Idp va tuyen chpn cdc ch&ng xg khuan tir ritng ngap man Cdn Gi& co kha nang sinh tong hap chat khdng ndm Fusarium sp.
Tir 5 mau dat liy tai RNM Can Gio, chiing t6i da phan lap dugc 55 kieu khuan lac xa khuan (tam gpi la chiing) khac nhau dugc ki hieu tu Fl den F55.
Chung toi tien hanh phan nhom cac chiing xa khuan d\ra vao cac dac diem sau:
mau sic k h u ^ lac, mau sic mep khuan lac, hinh dang mep khuan lac, sac to tan. 55 chiing xg khuan dugc phan thanh 3 nhom nhu sau:
f Nhom 1: Trang, g6m 19 chiing (34,5%).
+ Nhom 2: Xam - nau - den, g6m 25 chiing (45,5%).
+ Nhom 3: Vang nhat - vang - vang nau, g6m 11 chiing (20%).
Ti^n hanh tuyen chpn so bp cac chung xa khuin co kha nSng sinh t6ng hgp chat khang nim Fusarium sp. theo phuang phap ciy cham diem tren MT thach cua Crawford (1993). Ket qua cho thay 11 trong s6 55 chiing xa khuan phan lap dugc co
Tap chi KHOA HOC DHSP TPHCM 56 51 nam 2013
kha nang sinh tdng hgp chat khang nam Fusarium sp. (20%). Trong do: 4 chiing co hoat tinh khang nam Fusarium sp. manh (36,36%); 6 chiing cd boat tinh khang nam trung binh (54,54%) va 1 chung cd kha nang sinh tong hgp chit khang nim ydu (9,1%).
Chiing xa khuan F46 co kha nang sinh tdng hgp chat khang nam Fusarium sp. manh, cd tiem nang ttong viec bao ve cay trdng trudc su gay hai cua nam Fusarium sp. do do chung tdi chgn chung x^ khuan F46 de tiep tuc cac nghidn cihi.
Hinh 3.1. Khd ndng sinh long hpp chdt khdng ndm Fusarium sp.
ciia chiing xg khudn F46 3.2. Bac diim hinh thdi cua chung xa khudn F46
Tren MT Gause I sau 7 ngay ciy chim dilm, xa khuin tag thanh nhflng khuin lac trdn cd dudng kinh 9 mm. MJt ttdn khuin lac cd ciu tnic td ong mau nau, khuin ti khi sinh phat ttien theo hinh phdng xa va tao thanh cac vdng ttdn dong tarn bao ben ngoai.
Mep khuan lac mau tring, rang cua. Mat dudi khuin lac phing, khuin ti ca chit co mau trang, chinh gifla co mau vang nhat.
Hinh 3.2. Dgc diim hinh thai khudn lgc cua chung xg khudn F46
Sau 3 ngay nudi ciy ttong dia petti bing phuomg phap xe ranh tiiach, quan sat dudi kinh hien vi d do phdng dai xlOOO, khuin ti khi sinh khdng cd vach ngan, cuong sinh bao tii dang xoin Id xo. Bao tir dugc hinh thanh tren sudt chilu dai cua cudng sinh bao tir tao thanh chudi bao tii.
Hmh 3.3. Hinh thdi he sen khudn ti (a) vd cudng sinh bdo tic (b) eda chimg xg khudn F46 (xlOOO)
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Tap chi KHOA HOC DHSP TPHCM Hodng Thi Hdng va tgk
3.3. Khao sdt khd ndng sinh enzyme ngogi bdo
Theo Ismet Ara va cgng su (2012) [9], Humilton - Miller J.M (1973) [8] kha nang d6i khang ciia xa khuan voi nam dua tren kha nang sinh t6ng hgp hai nhom hgp chat sinh truang thii cap. Mgt la, khang sinh nhom polyen co tac dung pha v5 tinh tham thau ciia mang te bao chSt, dan den phd v6 chuc nang thdm chgn loc ciia mang te bao nam. Hai la, enzyme chitinase, glucanase pha vo thanh te bao cOa nam. Ngoai ra, enzyme ngoai bao con giiip xa khuan co kha nang tan dung nguon dinh du&ng ngoai MT cho sinh truong va phat trien [2]. Nham muc dich xac dinh ca che khang nam, ciing nhu kha nang sinh cac enzyme ngoai bao ciia chung xa khuan F46, chiing toi tien hanh khao sat kha n^ng sinh enzyme ngoai bao amylase, protease, cellulase va chitinase ciia chving xa khuan F46.
Bdng 3.1. Khd ndng sinh enzyme ngogi bdo cua chung xg khudn F46
Enzyme Amylase Protease Cellulase Chitinase
Birong kinh vong phan giai (D - d, cm) 0,73 + 0,12 0,27 ± 0 , 1 2 0,00 ± 0,00 0,47+ 0,25
Ket qua tren cho thay chiing xa khuan F46 co kha nang sinh cac enzyme ngoai bao amylase, chitinase va protease. Ket qua nay phii hgp voi quan diem ciia Nguyen Lan Dung (2000) [2] vd Bien VSn Minh (2006) [4] cho ring xa khuan co kha nang sinh nhieu loai enzyme ngoai bao. Kha nang sinh nhieu loai enzyme ngoai bao giiip chung xa khuan F46 tan dung dugc nhieu nguon dinh dudng trong MT de sinh truong va phat trien. Tuy nhien kha nang sinh cac enzyme ngo^i bao tren deu 6 miic yeu. Chimg x^
khudn F46 co kha ndng sinh t6ng hgp enzyme chitinase, co the noi kha nang sinh enzyme chitinase da gop phan tang kha nang khang nam ciia chung xa khuan F46.
3.4. Khdo sdt cdc dieu kien lin men dnh hudng den khd nang sink tong hgp chdt khdng nam Fusarium sp. cua chung xg khudn F46
3.4.1. Ufa chgn moi trucmg len men thich hap
Moi truong dong vai tro het sue quan trpng d6i voi su sinh truang va phat trien cua VSV. Chung t6i tien hanh len men tren 4 MT: Gause I, Gause II, Emerson va ISP4.
KSt qua dugc the hien a Bang 3.2.
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Bang 3.2. Hogt tinh khdng ndm Fusarium sp. cua chung xg khudn F46 tren cdc moi tru&ng len men khac nhau
STT 1 2 3 4
Moi trirdng Gause I Gause II Emerson ISP4
Ho^t tmh khang nam Fusarium sp. ( D - d , cm)
2,0'± 0,20 2,1 "±0,31 2,6''± 0,10 1,8'±0,25 Ghi chu: a, b, c chi su khac biet y nghTa vl thdng kd.
KSt qua tir Bang 3.2 cho thiy chimg xa khuan F46 sinh trudng va sinh tdng hgp chat khang nam Fusarium sp. tren ca 4 MT. Tuy nhidn, chimg xg khuan the hien kha nang sinh tdng hgp chat khang nim Fusarium sp. tdt nhit ttdn mdi tnrdng Emerson la MT giau djm hflu co. MT Emerson cd ngudn dam da dang, didu nay cho thiy chiing xa khuan F46 cd kha nang tan dung dugc nhidu ngudn dinh dudng khac nhau va chung xa khuan nay cung tham gia tich cue vao qua trinh chuydn hda vjt chit cua RNM. Ben canh do, thanh phin cua MT Emerson tuong ddi don gian so vdi cac MT con lai la mdt uu the khi tien hanh cac thi nghiem vd sau.
• I i^
Hmh 3.4. Hogt tinh khdng ndm Fusgrium sp. cug chiing xg khudn F46 tren MT len men khac nhau
3.4.2. Khdo sdt dnh huang ciia nguon nitrogen len khd ndng sinh ting hap chdt khdng ndm Fusarium sp. cua chiing xg khudn F46
Chung tdi tiln hanh nudi xa khuin F46 trong binh tam giac 250 ml chiia 50 ml MT Emerson dich thi va thay N tdng sd (peptone, cao nim men, cao thit) lin lugt bing peptone, cao thit, cao nim men, NH4NO3, NaNOj vdi lugng tuong duong. Kit qua dugc thi hidn d Hinh 3.5.
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Tap chi KHOA HOC DHSP TPHCM Hodng Thi Hong va Igk
~ V- -
f i l l B 1 dB m E
Peploiie Caotlut Caojiaiii NH4N03 NaN03 Peptone + men Cao tliit + Cao nam Nguon nitrogen "'^"
Hinh 3.S. Biiu do dnh huang cua ngudn nitrogen len khd ndng smh tdng hap chdt khdng ndm Fusarium sp. cua chung xg khudn F46
Qua bieu do cho thay chiing xa khuan F46 the hien kha nang sinh t6ng hgp chdt khang nam Fusarium sp. o tdt ca cac ngu6n N khac nhau. Voi ngu6n N huu co chiing xa khuan F46 the hien kha nang sinh tong hgp chat khang ndm t6t hon so voi khi sir dung nguon N vo ca. Tir ket qua tren chiing toi tiep tuc khdo sat chung x^ khuan F46 tren MT Emerson voi nguon N g6m cao ndm men, cao thit, peptone.
3.4.3. Khdo sdt dnh huang cita nguon carbon len khd ndng sinh tdng hap chdt khdng ndm Fusarium sp. cua chiing xg khudn F46
Chiing t6i tien hanh nu6i xa khuan F46 trong binh tam giac 250 ml chira 50 ml MT Emerson dich the va thay glucose ldn lugt bang maltose, sucrose, tinh bpt, lactose voi lupng tuong duong. Ket qua dugc the hien qua Hinh 3.6.
IjJjJ^
Tuili bot Suci o Lactose Ghicose
Hinh 3.6. Bieu do dnh huang cita nguon carbon len khd ndng sinh tdng hap chdt khdng ndm Fusarium sp. cua chitng xg khuan F46
Qua bieu do chiing toi nhdn thdy chiing xa khuan F46 the hien kha ndng sinh tong hgp chat khdng ndm tren tdt cd cac ngu6n C khac nhau. Di6u nay cho thdy chiing co kha ndng sii dyng nhieu ngu6n thiic an khac nhau do do co kha nang phan b6 rgng rdi trong ti^ nhien. Tuy nhien, chiing xa khuan F46 the hien khd nang sinh t6ng hgp chat khang ndm Fusarium sp. t6t nhat khi su dung ngu6n C la maltose. Chiing toi tiep tyc
Tap chi KHOA HOC DHSP TPHCM Sd 51 nam 2013
khao sat cac dilu kien tilp theo tren MT Emerson tiiay thi glucose bing maltose vdi lugng tuong duong (sau day ggi la MT E-1).
3.4.4. Khdo sdt dnh hudng cua pH ban ddu trong moi truang len men den khd ndng sinh ting hap chit khdng ndm Fusarium sp. ciia chimg xg khudn F46
Chiing tdi tiln hanh ldn men ttong binh tam giac 250ml chira 50ml djch MT E-I, dilu chinh pH theo cac gia ttj lin lugt la 4, 5, 6, 7, 8 (Gia trj pH 6 la gia tti ban diu cua
MT). Kdt qua dugc the Mend Bang 3.3. ^ Bdng 3.3. Anh hudng ciia pH ban ddu trong MT len men
den khd ndng sinh long hpp chdt khdng ndm Fusarium sp.cda chung xg khudn F46 STT
1 2 3 4 5
pH 4 5 6 7 8
Ho^t tinh khang nam Fusarium sp. (D - d, cm) 1,7" ± 0,00
2 , 2 ' ! 0,06 2,3"+ 0,06 1,9'+ 0,06 l,9'± 0,12 Ghi chu: a, b, c chi su khac biet y nghia vd thdng ke
Chiing xa khuin F46 thi hien hoat tinh khang nim Fusarium sp. d ca pH acid, trung tinh va kilm (pH 4 - 8). Chiing xa khuan nay thi hien kha nang sinh tdng hgp chat khang nam manh nhat d pH 5 - 6.
3.4.5. Khdo sdt dnh hu&ng ciia nong do muoi len khd ndng sinh tong hap chdt khdng ndm Fusarium sp. cua chitng xg khudn F46
Chimg xa khuan F46 dugc phan lap tit dat RNM do dd do man ciia MT anh hudng den kha nang sinh tdng hgp chit khang nim. Dl xac dinh nong do mudi thich hgp cho xa khuin F46 the hien kha nang sinh tdng hgp chit khang nim Fusarium sp.
manh nhit, chiing tdi tien hanh nudi xa khuan ttong binh tam giac 250mi chiia 50ml MT E-1 va dilu chinh ndng do NaCl theo cac gia tti lan lugt la 0%, 1%, 2%, 3%, 4%, 5% (0% la ndng dd NaCl cua MT ban diu). Kit qua dugc the hien d Hinh 3.7.
Hinh 3.7. Do thi dnh hucmg cua ning do NaCl len khd ndng sinh ting hpp chdt khdng ndm Fusarium sp. cita chitng xg khudn F46 66
Tap chi KHOA HOC DHSP TPHCM Hoang Thi Hong va tgk
Do man trung binh cua .RNM Cin Gid la 1,8 - 2,0%. Chiing xa khuin F46 cd kha nang sinh chit khang nim frong gidi hjn dp man rdng 0 - 5% va cd till sinh tdng hgp chit khang nim ngay ca d do man cao hon do man ttimg binh ciia RNM Cin Gid. Tuy nhien chimg xa khuin F46 till hien kha nang sinh tdng hgp chit khang ndm Fusarium sp. tdt nhit d ndng do NaCl 1%.
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Hinh 3.8. Khd ndng sinh tdng hap chat khdng ndm Fusarium sp. ciia chung xg khudn F46 a ndng do mudi khac nhau
Tir ket qua tren chiing toi tiep tuc khdo sat chung xa khuan F46 tren MT E-1 bo sung 1% NaCl (sau day gpi la MT E-2).
3.4.6. Khdo sdt dnh huang cua nhiet do len khd ndng sinh tdng hgp chdt khdng ndm Fusarium sp. cua chung xg khudn F46
Nhi§t dp CO anh huong sdu sac den qua trinh trao d6i chat cita VSV, chiing toi tien hanh khao sat kha nang sinh t6ng hgp chdt khang ndm Fusarium sp. ciia chiing xa khudn F46 o cac miic nhiet dp 25°C, 30''C va 3 5 ^ . K^t qud dugc the hien qua Bang 3.4.
Bdng 3.4. Anh huang ciia nhiet do len khd nang sinh tdng hgp chdt khdng ndm Fusarium sp. ciia chung xg khudn F46 STT
1 2 3
Nhift do 25°C 30°C 35°C
Hoat tinh khang nam Fusarium sp.
(D -d, cm) 2,6"± 0,06 2,l''±0,17 0,0'± 0,00 Ghi chii: a, b, c chi su khac biet co y nghia thong ke.
Chiing x9 khuan F46 the hipn kha ndng sinh tong hgp chdt khang nam o khoang nhi?t dg tu 25°C d6n 3 0 ^ va toi uu a nhi?t dp 2 5 ^ , la nhiet do tuong img voi nhiet do
Tap chi KHOA HOC DHSP TPHCM So 51 nam 2013
trung binh cua RNM Cin Gid (25,8°C) va chiing xa khuin F46 mit boat tinh d nhiet dp 35°C.
3.5. Xac dinh dgng thai qud trinh lin men
D I CO dugc mgt cai nhin khai quat vl cac giai doatn sinh tiudng, phat trien va kha nang sinh tdng hgp chit khang nim Fusarium sp. ciia chiing xa khuin F46, chiing tdi tiln hanh nghien ciiu ddng thai qua ttinh ldn men ciia chiing nay. Chung tdi tien hanh nudi chiing xa khuin F46 trong binh tam giac 250ml chiira 50ml MT E-2. Sau 24 gid, 48 gid, 72 gid, 84 gid, 96 gid, 108 gid, 120 gid, 132 gid, 144 gid, 156 gid, 168 gid xac dinh cac chi tieu sau: Kha nang sinh tmdng bing phuong phap xac dinh ttgng lugng khd tuyet ddi, kha nang sinh tdng hgp chit khang nim Fusarium sp. bang phuang phap khoan Id thjch, su thay ddi pH ttong binh len men.
Ddng tiiai qua ttinh len men cua chung xa khuin F46 dugc thi Men d Bang 3.5 va Hinh 3.9.
Bdng 3.5. Dong thdi qud trinh len men cua chiing xg khudn F46 STT
1 2 3 4 5 6 7 8 9 10 11
Thdi gian (gio')
24 48 72 84 96 108 120 132 144 156 168
Hoat tinh khang nam Fusarium sp. (D-d, cm)
1,6'"±0,18 1,8"° ±0,22 2,1**0,36 2,58 ±0,21 2,2"± 0,22 2 , l " ± 0,22 2,0" ±0,32 1,9'" ±0,23 1,9" ±0,21 1,5'±0,17 1,2" ±0,23
Sinh kh6i (g/l) 1,33"±0,18 1,87"=±0,02 2,13"'±0,02 2,33" ±0,05 2,07=" ±0,03 2,00"'±0,22 1,93"° ±0,00 1,73" ±0,11 1,73" ±0,22 1,33"±0,01 1,33"±0,02
pH 5,48" ± 0,23 7,40'" ±0,12 7,49" ± 0,12 7,29"± 0,12 7,44* ±0,12 7,47" ± 0,20 7,41'" ±0,23 7,42°" ±0,12 6,81" ±0,12 7,42'" ±0,23 7,40"" ±0,23 Ghi chii: a, b, c, d, e, f, g, h chi su khac biet cd y nghia vd thdng ke.
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Tap chl KHOA HOC DHSP TPHCM Hodng Thi Hdng va tgk
Thm gian (h)
- ^ " I ) - d i c i i i i — Sjiiliklionii ll pH
Hmh 3.9. Bd thi ddng thdi qud trinh len men ciia chiing xg khudn F46 Chiing toi nhdn thdy t6c do sinh truong ciia chiing xa khudn F46 tang ddn sau 24 gio nu6i cdy va sinh khoi dat cue dai sau 84 gio, sau do sinh khoi giam ddn do cac ngu6n dinh duong trong MT can kiet. Chiing xa khuan the hien khd nang sinh tong hgp chat khang ndm Fusarium sp. sau 24 gio va tang o cac miic thai gian sau do. Trong khoang thoi gian tir 72 gio den 120 gio chiing xa khudn the hien hoat tinh khdng ndm Fusarium sp. manh va manh nhdt sau 84 gio. Sau 132 gio hoat tinh khdng ndm giam xu6ng miic trung binh va gidm nhanh sau 156 gia.Sinh kh6i va khd nang khang nam Fusarium sp. dat gia trj cue dai 6 ciing mgt thoi gian thudn lgi cho qua trinh thu sinh khoi de trgn vao dat khi khao sat khd ndng doi khang nam Fusarium sp. cOa chiing xa
• khudn F46 tren cdy tr6ng.
pH ciia MT bat dau tang sau 24 gio nu6i cdy va gdn nhu cdn bang 6 khoang thai gian til 48 gio den 132 gio. pH gidm xuong khi 6 thai gian 144 gio va tdng tra lai sau do.
4. Ket lu^n va ki^n nghj 4.1. KSt lugn
Qua qua trinh tien hanh de tai, chting toi da thu dugc nhiing ket qud nhu sau:
• Phdn lap dugc 55 chiing x? khudn khac nhau va phan thanh 3 nhom:
+ Nhom 1: Trang, chiem 19 chiing (34,5%).
+ Nhom 2: Xam - nau - den, chiem uu the voi 25 chiing (45,5%).
+ Nhom 3: Vang nhgt - vang - vang nau, chiem 11 chung (20%).
11 trong so 55 chiing xa khudn phdn lap c6 hoat tinh khdng ndm Fusarium sp.
chiem 20%. Trong do, 4 chiing co hoat tinh khdng ndm manh, 6 chiing co hoat tinh khang ndm trung binh va 1 chung co hoat tinh khang ndm ylu.
• M6 td dugc ddc diem hinh thai ciia chiing xa khudn F46
T?p chi KHOA HOC DHSP TPHCM S6 51 nam 2013
+ Hinh thai dai the: Khuan lac gdm 3 lop, lap ngoai la nhimg khuan ti khi sinh phat triln theo hinh phdng xa va tao thanh cac vdng trdn ddng tam mau trang, lop trong cd ciu true xdp hon, Idp giOa co ciu tinic td ong mau nau.
+ Hinh thai vi thi: khuin ti khi sinh khdng cd vach ngan, cudng sinh bao tit dang xoin Id xo. Bao tir dugc hinh thanh tten sudt chilu dai cua cudng sinh bao tii tao thanh chudi bao tir.
• Chung xa khuan F46 khang nam Fusarium sp. tdt (D-d = 2,2cm > 2cm) tten MT Emerson.
• Chung xa khuan F46 the hien kha nangsinh tdng hgp chat khang nam tdt nhat vai ngudn N hiru eg, da dang (4g cao thit + 4g peptone + Ig cao nam men/1 lit nudc cat), ngudn C la maltose, pH = 5 - 6, ndng do NaCl la 1%, nhiet do 25°C).
• Chiing xa khuin F46 sinh trudng va thi hien kha nang sinh tdng hgp chat khang nam Fusarium sp. cue dgi sau 84 gid.
4.2. Kien nghi
Djnh danh din loai chiing XEi khuin F46.
Xac dinh tinh chit li, hda cua chit khang nim Fusarium sp. sinh tdng hgp tir chung xa khuin F46.
Tien hanh thi nghiem xac dinh kha nang khang nim Fusarium sp. tir djch nudi ciy xa khuan F46 ttong didu kign in vivo.
TAI LIEU THAM KHAO
1. Lester W. Burgess, Timothy E. Knight, Phan Thuy Hiln (2009), Cdm nang dodn bfnh cdy trong a Viet Nam, Trung tam Nghien ciru Ndng nghiep Qudc t l Austtalia, tt.126-150.
2. Nguyen Lan Dung (2000), Vi sinh vdt hoc, Trung tam Cdng nghe Sinh hge, Dai hge Qudc gia Ha Ndi, tt.96-147.
3. Bill Thi Viet Ha (2006), Nghien cuu xg khudn sinh chdt khdng sinh ching ndm gdy benh thifc vdt a Viet Nam, Luan an Tiln si Sinh hge, Trudng Khoa hge Tu nhien, D?i hoc Qudc gia Ha Ndi, tt.3-20.
4. Biln Van Minh (Chu bidn), Kidu Hthi Anh, Pham Nggc Lan, Pham Hdng Son, Pham Van Ty, Nguyin Thi Thu Thiiy (2006), Vi sinh vdt hoc, Nxb Dai hge Hul, 321 tr.
5. Trin Thi Thanh (2007), Cong nghe vi sinh, Nxb Giao due, tr.I20-137.
6. Pham Thi Thuy (2010), Gido trinh cong nghe sinh hpc trong bdo ve thifc vdt, Nxb Giao due Viet Nam, tt.24-25.
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Tap chi KHOA HQC OHSP TPHCM Hodng Thi Hong vd tgk
7. Crawford DL, Lynch JM, Whipps JM, Ousley MA (1993), "Isolation and characterization of actinomycete antagonists of a fungal root pathogen". Applied and Environmental Microbiology, 59(3), pp. 899-905.
8. Humilton - Miller J. M. T. (1973), "Chemistry and biology of the polyene macrolide onixhiotACs", Bacteriological reviews., 37(2), pp. 166-196.
9. Ismet Ara, Bukhari N. A, Perveen K, Bakir M. A (2012), "Antifungal activiti of some actinomycetes isolated from Riyadh soil, Saudi Arabia: An evaluation for their abiliti to control Alternaria caused tomato blight in green house pot trial", African Journal of Agricultural research, Vol. 7(3), pp. 2042-2050.
(Ngty Toa soan nh$n tfopc b^i: 21-6-2013; ngdy phin bi$n tJant} gii: 07-8-2013;
ngiy chip nhin ding: 30-8-2013)
