CHIN LC TRUYN THNG PHNG CHNG BNH VIRUT HI CY TRNG

1. LCH S NGHIN CU BNH VIRUTS HI CY TRNG

* S pht hin ra virus hi cy trng A. Mayer (1886) pht hin s ly lan ca bnh khm thuc l nhng ng coi l mt bnh vi khun D.Ivanopski (1892) sau khi pht hin bnh ng cho rng l mt cht c. Mi ti M.Bayerinck (1898) mi xc nh virus l mt nguyn nhn gy bnh mi nh b hn vi khun. Cc cng trnh ca A.Mayer, D.Ivanopski, M.Bayerinck v sau ny l ca Loeffler, Frosch, 1898 m u cho mn virus hc thc vt v sau ny l c mn virus hc ng vt v ngi pht trin v tr thnh mt ngnh khoa hc ln hin nay trong sinh hc hin i ca th gii. Cc cng trnh nghin cu virus tip sau dn xc nh chnh xc cc virus hi thc vt. Virus (TMV) ln u c quan st thy vo nm 1931 - 1939 trn knh hin vi in t u tin. T vic nghin cu hnh thi hc virus c pht trin nhanh chng ngi ta pht hin ra hnh thi nhiu virus. Phng php huyt thanh c s dng trong nhng nm 30 to chuyn bin ln trong nghin cu virus. Tuy vy, sau nhiu nm s dng cc phng php quan st huyt thanh thng thng khng c hiu qu cao, phng php Latex cng khng khc phc c. Nm 1977, Clark v Adams ln u tin pht trin phng php ELISA chn on cc cy bnh virus - phng php ny thu c kt qu rt kh quan. n nm 1982 ngi ta s dng phng php DNA probe v phng php PCR (Polymeraza chain reaction). Phng php ny gip vic chn on virus thc vt chnh xc v nhanh chng trong trng hp cy ch c triu chng bnh rt nh hay bnh hon ton dng n cng c th pht hin c. Cc tin b trn y cng c ng dng nghin cu to cy sch, to ging chng bnh. Ngi ta s dng gen ho m v protein ca virus gy min dch v phng php Cross protection (bo v cho) c ng dng c hiu qu. Ngy nay, vi cc ng gp ca Frankin, M.V.H. Van Regenmortel, C.M. Fauquet,

D.H.L. Bishop v nhiu tc gi trong vic phn loi virus v nhiu nh khoa hc nghin cu sinh l, sinh ho, hnh thi, sinh thi hc, mn virus hc tr thnh mn khoa hc hin i v pht trin, tm ra nhiu quy lut v sinh hc mi.

2. NHNG THIT HI CA BNH VIRUTS CY TRNG.

2.1. Nhng thit hi chung ca bnh virus thc vt Bnh virus thc vt gy thit hi ln nht khng phi l cho cy trng b cht nhanh chng m chnh l chng lm cho cy b thoi ho, gim sc sng, dn tn li. cy lu nm mt s virus sau khi gy bnh nng trong ma khi c thi tit v nhit n ho, nhng khi nhit thp hay qu cao thng gy nn hin tng mt triu chng (latent periode) lm cho ngi sn xut b nhm ln, khng pht hin c cy b bnh v mc nguy him ca bnh, ch n lc no cy khng cn kh nng phc hi theo chu k bnh na, cy hon ton tn li, khi mi bit th qu mun. Virus cng c th gy nn nhng thit hi nng n v nhanh chng ngay trong v trng ca cc cy thng nm nh virus gy bnh la vng li bnh vng l la, bnh xon l c chua, bnh virus khoai ty, bnh khm sc l hnh ty. Bnh virus hi la, virus hi sn tng hu dit hng chc vn ha chu v chu Phi trong mt thi gian ngn cha ti 30 ngy t mt cnh ng xanh ti tr thnh vng a, cht li. Thit hi quan trng th hai ca virus l nh hng ca bnh ti phm cht ca cc sn phm nng nghip: ht la b bnh vng li thng b lp khng cho thu hoch, trong trng hp c thu hoch ht thng rt nh v ht go b en, khi n c v ng. Khoai ty b virus gy hi lm cho cy cn ci, l khm loang l, c khoai nh, hm lng tinh bt v cc cht dinh dng u thp. C trng hp bnh do mt chng c bit ca virus lm v qu, c c vt lot, bn gim gi tr thng phm, nh khi khoai ty b nhim mt chng virus Y. c chua b xon l qu b, mi kh v hoa rng; nng sut v phm cht u rt thp. Virus Tristeza hi cy cam ngt rt nng trn vng b bin a trung Hi, Trung M, ng Nam lm cho qu cam chn p v rng sm, qu cn non a vng v, nc cam nht khng mi, v. Bnh virus cn nguy him ch: virus k sinh bt buc trong t bo cy k ch v vy khi t bo b hu hoi, cht,

virus mi b mt hot tnh. Khi t bo non pht trin mnh, virus cng pht trin mnh, to ra nhng triu chng rt in hnh trn cy non hay b phn non ca mt cy. Chnh v vy khi nhn ging v tnh bng invitro, virus c kh nng ly lan rt ln trong vic pht trin cng ngh sinh hc v cc vng trng trt cng ngh cao. i vi nhng cy trng nhn ging v tnh nh cam, qut, khoai ty, khoai lang, sn nhn bng nui cy m, chit, ghp virus l nguy c hu dit rt ln i vi nhiu loi cy trng. Chng kh pht hin v kh loi tr. Chnh v vy, chng tr thnh mt k th nguy him ca cng ngh sinh hc. cc nc pht trin, vic tiu dit bnh hi, loi tr bnh hi, to cy sch v cy chng chu virus, phytoplasma, viroide l mt cng vic quan trng trong sn xut nng nghip. 2.2. Thit hi ca bnh virus Vit Nam. Vit Nam l mt t nc c chiu di gn 2000 km, kh hu nhit i gi ma. Trong nm c mt ma ng lnh pha bc. Chnh v vy, cy trng v thm thc vt Vit Nam bn ma xanh ti. y l iu kin bnh virus Vit Nam rt phong ph v s lng v chng loi. Bnh gy ra rt nhiu thit hi nng n cho sn xut. V d: bnh vng li tn ph hng trm ngn ha la min ni, trung du v ng bng hai min Bc, Nam. Bnh vng l cam, qut hu dit nhiu vn cam, nhiu vn khc trong tnh trng thoi ho, gim nng sut nghim trng. Cc cy bng, h tiu, ca cao u b bnh gy hi. Cy h c nh thuc l, c chua, khoai ty, cc loi rau bu, b, cc cy h u: u tng, u xanh, u en cc cy n qu nh chui, da, u .. u mc bnh. Ngoi cy lng thc, cy cng nghip, cy n qu.v.v. virus cn hi cc loi cy thuc, cy hoa, cy cnh c th ni rng virus l mt loi bnh nguy him, ph hoi hu ht cc loi cy trng. Bnh gy nn s thoi ho dn n tn li cy trng, thm ch c th hu dit nhanh chng nhng din tch ln trong sn xut trng trt. Bnh khng nhng lm gim nng sut cy trng m cn lm gim phm cp ca sn phm. V vy y l mt loi bnh gy hi ton din v rt nguy him cho ngnh trng trt nc ta cng nh trn th gii.

3. C TNH CHUNG CA VIRUTS HI CY TRNG.

3.1. Virus hi thc vt l nhng nucleoprotein rt nh b

Nhng virus dng cu nhm Luteo virus kch thc ch t 23- 24nm. Nhng virus dng cu nhm Ilavirus c kch thc bin ng t 26 - 35 nm. Hn mi nhm virus khc cng c kch thc bin ng trong khong 29nm, 30 34nm. Virus ln nht dng cu l Tomato spotted wilt cng ch c ng knh 80nm. Nhm Rhabdoviridae l virus dng vi khun to nht (135 - 380 x 45 95nm). Virus di nht l cc virus dng si nhm Clostero virus di 2000 x 12nm. Chng nh b nh vy nn vic tm kim pht hin chng i hi phi c nhng phng php c bit. 3.2. Virus k sinh mc t bo v chng c kh nng nhn ln trong t bo Ngi ta pht hin thy virus nhim cc c th nh b nh mycoplasma, vi khun, nm cho ti cc thc vt thng ng, ngi, ng vt Theo cc ti liu, hin nay ngi ta bit ti 2000 loi virus gy bnh cho cc sinh vt trong c 1/2 ( khong hn 1000 loi) l cc virus hi thc vt cha k n cc chng loi ca chng. 3.3. Virus c cu to rt n gin, chng c 2 thnh phn chnh l protein v axit nucleic. Li axit nucleic bn trong cbao bng mt lp v protein (capside). Thng axit nucleic ca virus gy bnh cy hu ht l cc virus c genom l ARN chui n (+), ch c mt s t genom ARN l mch kp v c rt t. Khong hn 25 loi l virus c li ADN mch kp. Virus gy bnh cy thng ch c 1 loi protein. 3.4. Virus c kh nng hot ng chng chu vi cc iu kin ngoi cnh: xc nh kh nng chng chu ny bng 3 ch tiu: thi gian tn ti dng dch, ngng pha long v nhit lm mt hot tnh (Q10 ). 3.5. Virus cn c kh nng bin d: virus d dng to thnh chng (strain) mi khi cy k ch v mi trng sng thay i v virus c th mt hot tnh (ni cch khc l virus cht). Virus c nhiu c tnh khc, vi cc nguyn nhn gy bnh khc khng phi ch do chng nh b m do cu trc vt l, cu to ho hc, cch xm nhim, sinh sn v di chuyn trong t bo cy k ch, triu chng to thnh trn cy k ch cng rt khc bit. Tuy nhin, cho ti nay tri qua hng

trm nm khi virus c pht hin trn th gii cho ti nay cc nh virus hc vn cha i n mt kt lun chnh xc: virus c phi l mt sinh vt theo ng ngha ca n hay khng Bn cnh nhng c im ging nh mt sinh vt nh c im xm nhim v gy bnh, s di truyn v bin d, c b mt hot tnh (cht). Virus li c th tch ARN v protein ring, ri ghp genom (ARN) ca n vi mt v protein khc, virus li tr li hot ng bnh thng. Virus cn c th to thnh dng kt tinh trong t bo cy. Nhng c im ny hon ton khng phi l c im ca mt c th sng. Tuy nhin, cho n nay virus vn c xp vo nhm cc vi sinh vt gy bnh cho cy, cho ngi v cho gia sc, v chng c kh nhiu c im ging mt sinh vt nh b, s phn loi ny ch mang tnh tng i.

4. TRIU CHNG BNH VIRUTS HI CY TRNG.

Vic phn loi triu chng bnh virus hi thc vt c ngha quan trng trong chn on, phng tr v nghin cu bnh hi. Tuy nhin, s phn loi bnh ch c tnh cht tng i v din bin triu chng bnh rt phc tp v cch phn loi cn tu thuc vo quan im ca mi tc gi khc nhau. Qua nghin cu bnh virus thc vt, nhiu tc gi chia bnh thnh cc nhm sau: 4.1. Khm l Bnh virus thng xm nhim vo l cy gy ra hin tng khm l, loang l, ch xanh m, ch xanh nht, ch bin vng. y l triu chng ph bin nht vi hu ht cc bnh virus hi cy. C th nu v d mt s bnh sau: virus khm thuc l, khm l t, khm l da chut, khm l u, khm l khoai ty. a) Khm m cht c hnh nhn Nh bnh m hnh nhn cy u , cy mn, cy thuc l, cy hoa cm chng b) Hin tng gn l cht, sng gn, bin dng, Virus khm l khoai ty (dng khm nhn) to ra cht gn cy khoai ty, gn l trong cy thuc l (bin dng gn do virus qun l gy ra) c) Hin tng khm l, ln cy

Khm l km theo cy ln cng l mt dng bnh rt ph bin cy bnh virus: nh bnh khm ln cy ng, bnh vng ln cy la, xon ln cy bng, 4.2. Bin dng: nh xon l c chua, cun l khoai ty, xon l h tiu, xon l t, khm nhn l khoai ty,Ngoi bin dng l cn hin tng bin dng c qu. Nh bnh m ho c chua, bnh vng ln khoai ty, bnh virus to, nho, mn cng gy bin dng qu. 4.3. Bin mu: nh bin vng la, vng l cy cam, vng l cy u, 4.4. Hin tng tn li: cy ci cc, ln, mc tng bi nh bnh ln bi cy lc, bnh la c, bnh Tristeza cam, bnh chm ngn chui. 4.5. Gy vt cht thn cy: Bnh vng l cam, gy ra vt lm thn cy cam chanh v virus sng cnh to. Cch chia nhm c trnh by trn c ngha nhn bit bnh nhanh thng qua vic quan st thc t. Mt s tc gi chia triu chng bnh thnh cc nhm: Nhm bnh nhim h thng (nhim ton cy) v nhm bnh nhim b phn (gy vt cht cc b). Cch chia nhm ny gip cho vic chn on bnh hi s b lc u. Tuy nhin, trong trng hp no c virus va gy cc vt cht va nhim h thng. Vic phn nhm vn ch c tnh cht tng i. Tuy nhin, y l cch chia nhm c gi tr cao hn cch chia nhm theo triu chng n thun. 4.6. Tnh chng chu ca virus thc vt Virus l mt k sinh rt nh b, k sinh tuyt i trong t bo thc vt. Trong trng hp virus b tch ra khi t bo, nm trong dch cy, chng s rt kh tn ti. Nghin cu vn ny c ngha quan trng v nhm virus truyn c hc mun truyn bnh phi sng mt thi gian trong dch cy mi c th truyn n mt t bo sng khc ca thc vt. Nhiu tc gi i n nhn xt: cc yu t - s ko di i sng ca virus trong mt dch cy b xi ho do mi trng, tc ng ca cc mc nhit cao, thp khc nhau, kh nng dch cha virus b pha long do ma, mi trng, c nh hng rt r rt n vic bo tn sc ly bnh ca virus qua mt k ch mi. Vi nhn xt trn cc nh virus hc kho st kh nng chng chu ca virus vi mi trng bng 3 th nghim n gin:

+ Thi gian tn ti ca virus trong dng dch: ly 10 hay 20 ng nghim kh trng c nt kn, iu kin nhit trong phng th nghim v quy nh thi gian ly tng ng nghim ra ly bnh cho cy kho. Thi gian no ko di nht m dch cy vn cn kh nng ly nhim bnh ngi gi l thi gian tn ti trong dng dch ca virus. V d: - Virus Y khoai ty (PVY): 15 pht - 1 gi - Virus khm l ma (SCMV): 1 ngy - Virus khm thuc l (TMV): 1 thng - Virus X khoai ty (PVX): 1 thng + Ngng pha long: cng b tr th nghim tng t v ly bnh cho cy kho cc mc pha long: 1 gi nguyn dch chit t l cy nhim virus, pha long 1/2, 1/4,...1/8...1/16...1/32...1/64...1/128... ngng pha long s l mc pha long cao nht m dch cy cha virus cn gi c kh nng ly bnh. Sau mc pha virus khng ly bnh c na. V d: - Virus Y khoai ty (PVY) l 10-2 - Virus X khoai ty (PVX) l 10-5 - Virus khm thuc l (TMV) l 10-6 + Nhit lm mt hot tnh (Q10): l nhit trong bnh un cch thu (c nh nhit trong 10 pht) m nhit virus bt u mt hon ton kh nng ly bnh. V d: - virus Y khoai ty (PVY) Q10 = 520C Virus X khoai ty (PVX) Q10 = 720C - Virus khm thuc l (TMV) Q10 = 930C - 960C tu theo chng Tu theo chng virus m Q10 c th dao ng 1 vi . V d virus TMV c th c chng c Q10 bin ng t 93 0C - 960C.

5. HNH THI V CU TO CA VIRUTS THC VT.

5.1. Hnh thi Virus thc vt v virus hi sinh vt ni chung c hnh dng v kch thc rt a dng. Chng c th c dng hnh gy ngn, hnh gy di, hnh cu, hnh khi a din, hnh si ngn, si di, hnh vi khun v nhiu dng khc. a) Nhm virus hnh gy - Virus khm thuc l (TMV) kch thc 15 x 300nm - Virus khm l u h lan kch thc 46 - 200 x 22nm - Virus khm sc l la mch kch thc 100 - 150 x 20nm b) Nhm cc virus c hnh si mm - Virus X khoai ty (PVX) kch thc 480 - 580 x 13nm (si ngn) - Virus A khoai ty (PVA) kch thc 680 - 900 x 13nm (si di trung bnh) - Virus Tristeza hi cam, chanh v virus bin vng c ci ng c kch thc t 800 - 2000 x 12 nm. (si di nht) Thuc nhm Potex virus (d), Poty virus (e), Clostero virus (f). c) Nhm virus c cu to i xng dng hnh cu - Virus m cht l thuc l dng cu, ng knh 30 nm - Virus khm l da chut dng cu, ng knh 29 nm - Virus khm l sp l dng cu, ng knh 50 nm Thuc nhm Tymo virus (g), Cucumo virus (h), Caulimo virus (i). d) Nhm virus c cu to i xng hnh vi khun - Virus m cht vng rau dip, kch thc 300 x 52nm Nhm Alfaifa mosaic virus 28 - 58 x 18nm Thuc nhm Rhado virus (k).

e) Cc nhm virus c hnh dng khc: l nhm Germini virus v Tenui virus. 5.2. Cu to a) Cu to Bnh thng mi mt virus u c cu to t protein v axit nucleic, mt s virus c bit cn cha c polyamin, lipit hoc men c hiu (nh thc th khun Bacteriophage). T l axit nucleic v protein thay i vi mi loi virus khc nhau. Axit nucleic thng chim t 5 - 40%, cn protein nhiu hn thng chim t 60 95%, lng axit nucleic thp v protein cao c th thy cc virus c hnh si di tri li lng axit nucleic cao v protein thp c th thy cc virus c dng hnh cu. Trng lng ton c th ca virus cng rt khc nhau, t 4,6 triu n v trng lng phn t virus khm l c Brome, 39 triu virus khm l thuc l v 73 triu bnh virus gin thn thuc l,... b) Protein ca virus thc vt Cng c to thnh t nhiu axit amin nh alalin, acginin, sistein, glixin, lizin, lesin, fenilalamin, treonin, prolin, triptophan, tirozin, valin, axit asparagimic, axit alutamic,... Cc axit nucleic ca virus: ARN hay ADN quyt nh bn cht protein ca chng (thnh phn cu to, s sp xp,...). V d: virus khm thuc l (TMV) l virus c dng hnh tr ngn (hnh gy ngn) kch thc o c300 x 15nm. Cc phn t protein c sp xp theo hnh xon (l xo) bao quanh, mt chui cc axit nucleic (ARN). gia hnh tr c mt li rng. Cc phn t protein cng sp xp dng xon bao quanh gi l v (capside) ca virus. Lp v ca virus khm thuc l c 161/3 n v phn t protein cho mt vng xon, v virus c 130 vng xon. cc virus hnh si xon mm nh nhm Potex virus, nhm Poty virus, nhm Clostero virus,... s sp xp ca li axit nucleic (ARN) v v protein cng tng t virus khm thuc l. cc virus hnh cu nh nhm Luteo virus, nhm Cucumo virus,...cc phn t protein v axit nucleic sp xp i xng qua tm ca hnh cu ging nh mt khi a din. Cc virus c dng hnh vi khun, nh virus thuc h Rhaboviridae v

Caulimoviridae l nhng virus c cu to i xng qua trc xuyn tm. i vi mt s virus ADN thuc h Caulimoviridae protein c xp vng quanh, cn cc si ADN nm thnh tng vng khp kn gia (hnh dng cc virus c m t trn hnh). c) Axit nucleic ca virus thc vt Phn ln cc virus thc vt c cu to genom l ARN chui (+) v v bc ngoi l protein. Mt s t c ARN chui kp, khong 25 virus thc vt cha li ADN chui kp, ngc li virus ng vt th phn ln l virus c genom l ADN. C ARN v ADN u l nhng chui phn t di, cha hng trm hay nhiu hn l hng ngn cc n v nh c gi l nucleotit. Chui polynucleotit ny c phn t lng l 2,5.106 ( virus khm l thuc l). d) Th kt tinh ca virus Mt s virus trong iu kin nht nh ca mi trng c th to thnh tinh th. Nm 1935, W. M. Stanley tch c tinh th ca virus khm thuc l (TMV). Virus ch to thnh tinh th khi chng trng thi tnh (virion). Mt s virus to th kt tinh khi ta x l amonisunphat. Ngy nay, ngi ta c th to ra tinh th virus ngay trong ng nghim v bn cht ca hin tng ny l do tc dng ca cc lc ni kt gia cc phn t v ph thuc cu to l ho b mt ca cc vt th nh b khng phn bit l sinh vt hay phi sinh vt. Trong th nghim y hc ngi ta lm kt tinh virus gy bnh bi lit khi to ra im ng in trong ng nghim. Tinh th ca virus thc vt c quan st thy rt r di knh hin vi quang hc thng thng nht l cc virus thuc nhm Tabamo virus hay Poty virus,... Tuy nhin, s xut hin ca chng ph thuc vo tnh trng ca cy lc ly mu v iu kin mi trng. e) Chc nng axit nucleic v protein: trong cu to c th virus thc vt c nhng chc nng khc nhau: axit nucleic gi vai tr quyt nh tnh di truyn xm nhim v ly bnh ca virus thc vt, protein c tc dng bo v, bm gi v c vai tr quan trng trong khi virus truyn bnh qua mi gii truyn bnh.

6. S XM NHIM V TNG HP VIRUS MI.

6.1. S xm nhim ca virus Virus xm nhp vo t bo qua cc vt thng nh do sy st v nh s tip xc ca git dch cha virus hoc do c st tip xc gia l cy bnh, cy kho m virus xm nhp vo t bo. Virus cn c th truyn bnh trong trng hp mt ht phn hoa b nhim virus cri vo mt non thc vt. Trong m cy b nhim bnh virus di chuyn trong t bo cht ca t bo v c th i sang cc t bo khc thng qua cc si lin bo hay cc vt thng m ra vch t bo. 6.2. S ti sinh virus a) Khi nim S ti sinh (replication) hay sinh sn l s hnh thnh phn t virus mi t phn t virus ban u. Sau khi xm nhp vo t bo k ch, s ti sinh virus tri qua 4 giai on: 1. Tho v gii phng b gien virus 2. Tng hp protein virus 3. Tng hp b gien virus mi 4. Lp rp phn t virus b) c im chung S ti sinh virus, mc d khc nhau ty nhm, nhng u c c im chung sau 1. Virus s dng vt liu ca t bo k ch (amino acid, nucleotide) tng hp protein v acid nucleic ca chnh virus. 2. Virus s dng nng lng ca t bo k ch (ch yu di dng cc hp cht cao nng nh ATP) tng hp protein v acid nucleic ca chnh virus. 3. Virus s dng b my tng hp protein ca t bo k ch (ribosome, tRNA v cc enzyme lin quan) tng hp protein ca virus. Qu trnh tng hp s da

trn khun mRNA ca virus. Tt c virus thc vt s dng ribosome 80 S ca t bo k ch. 4. Hu ht cc virus thc vt tng hp 1 hoc 1 s enzyme cn thit cho qu trnh tng hp b gien virus. V d: a. Tt c cc virus RNA m ha RdRp (RNA-dependent RNA polymerase). RdRp l mt enzyme polymer ha v c chc nng tng hp RNA trn khun RNA. b. Cc geminivirus (c b gien DNA si vng n) m ha Rep (replication) protein. Rep khng phi l mt enzyme c chc nng polyme ha nhng c chc nng ct v ni cc phn t DNA virus trong qu trnh tng hp si DNA virus. Tm li, s ti sinh virus ph thuc hon ton vo b my tng hp protein v acid nucleic ca t bo k ch. S d nh vy l do virus ni chung v virus thc vt ni ring ch m ha mt s t gien; v d cc begomovirus ch m ha 5 8 gien, cc potyvirus ch m ha 10 gien. Do phi da hon ton vo vt cht ca t bo thc vt sinh sn, cc virus pht trin mnh trn cy non v t bo non trong mt cy. cc cy gi ci, qu trnh ny s chm li hay hu nh ngng hn. Chnh v vy, tui cy non v phn non ca cy l ni virus sinh sn rt mnh. Cc iu kin ngoi cnh nh: nhit qu cao, thp, pH ca mi trng, nh sng, ch dinh dng, chm sc. Mt cht c nhiu nh khoa hc xc nhn c bn cht protein tn l interferon c th sn sinh ra t bo k ch khi virus xm nhp. Vi nng thp khong mt phn triu gram c kh nng c ch sinh sn ca virus. Chnh v nhng l do trn bnh virus khng gy c tc hi hu dit ngay m thng gy thoi ho. S hu dit ch xy ra khi iu kin mi trng v cy bnh thun li cho virus sinh sn v ly nhim, nh trong cc trn dch ca bnh la vng li nc ta nhng nm 1960. 6.3. S di chuyn ca virus trong t bo cy. Virus xm nhp vo t bo cy, chng di chuyn theo dng t bo cht hoc c trng hp virus di chuyn theo cc dng nha nguyn v dng nha luyn ca cy, ln vo s di chuyn ca cc cht dinh dng, nc hay mui khong ca mch dn thc vt. Virus di chuyn t t bo ny qua t bo khc qua cc cu ni nguyn sinh mt cch chm chp. Quan st mt virus khoai ty xm nhp vo ngn mt l k ch di thp ca cy khoai ty non, G. Samuel ghi

nhn: sau 3 ngy virus mi nhim ht mt l n, sau 4 ngy mi nhim ht on gn ca l kp v mt phn on thn st gc, sau 5 ngy mi nhim ht dc theo gn chnh v mt l ngn (cc l khc cha h nhim virus). Sau 10 ngy mi nhim ht 2 l ngn, 1 thn chnh v l kp ni virus ly nhim vo u tin... ti 25 ngy sau virus mi nhim trn ton cy khoai ty b ly nhim. S dch chuyn ca virus trong ton cy to thnh mt cy bnh nhim h thng vi cc triu chng ton cy nh khm l, xon l, ln cy, ln bi... C trng hp virus cho ly nhim cc b trn l, to vt cht, khng ly lan ton cy thng gi l cy nhim bnh cc b: l dng vt cht hoi trn cy thuc l di, cy c c dc, cy cc bch nht... khi nhim cc virus TMV, PVX...

7. PHN LOI VIRUS THC VT.

Vic phn loi virus gy bnh c chn on theo U ban quc t v phn loi virus (International committee on taxonomy of viruses - gi tt l ICTV) da vo c im cu to, hnh thi ca virus cng nh mi quan h huyt thanh v cc c tnh khc nh c im truyn lan, ly nhim, phm vi k ch c bit l cc c im di truyn ARN v ADN. Tn gi ca virus hi thc vt quy nh dng ting Anh bao gm tn ca cy k ch chnh, triu chng bnh trn cy k ch v cui cng l t virus. V d: virus gy bnh khm thuc l - Tobaco mosaic virus - vit tt l TMV. Theo Agrios G.N, 1997 trong h thng phn loi tt c cc loi virus thc vt thuc ngnh virus (Kingdom: viruses). Trong ngnh tu theo cu to ca axit nucleic chng chia lm 2 nhm ARN virus v ADN virus da vo axit nucleic ca virus l ADN hoc ARN. Chng ti s dng bng phn loi theo Claude Fauquet (2001). 7.1. Nhm ARN virus a) Cc virus c ARN si n dng (ss ARN +) + ARN virus dng hnh gy: gm 32 loi - 1 ssARN (+) Ging Tobamovirus V d: Tobaco mosaic virus (virus khm thuc l)

- 2ssARNs Ging TobravirusV d: Tobaco rattle virus - 2-4 ssARNs: virus hnh gy truyn qua nm Ging Furovirus. V d: Soil-borne wheat mosaic virus (virus khm l la m c ngun gc t t). - 3ss ARNsS Ging Hordeivirus. V d: Barley stripe mosaic virus (virus khm sc nh la i mch) + ARN virus dng si mm: 280 loi - 1ssARN Carlavirus V d: Carnation latent virus (virus n hoa cm chng) Trichovirus V d: Apple cholorotic leafspot virus (virus m vng l to) PotexvirusV d: Potato virus X (virus khm l khoai ty) H Potyviridae - 1ssARN virus hnh si mm di Ging Closterovirus: V d: Beet yellows virus (virus vng l c ci ng) + ARN virus dng hnh cu: 165 loi - 1ssARN (+) H: Sequivirisdae Waikavirus. V d: Rice tungro spherical virus (virustungro dng cu hi la) H: Tombusviridae Ging: Tombusvirus V d: Tomato bushy stunt virus (virus chn ngn tn li c chua)

Ging Carmovirus V d: Carnation mottle virus (virus m l cm chng) Ging Machlomovirus V d: Maize chlorotic mottle virus (virus m vng ng) Ging Necrovirus V d: Tobaco necrosis virus (bnh m cht thuc l) Ging Luteovirus V d: Barley yellow drawf virus (virus vng ln) Ging Sobemovirus V d: Southern bean mosaic virus (virus khm l u) Ging Tymovirus V d: Turnip yellow mosaic virus (virus khm vng cy cci) - 2ssARN (+) H: Comoviridae Ging: Comovirrus V d: Cowpea mosaic virus (virus khm l cy u a) Nepovirus V d: Tobaco ringspot virus (virus m hnh nhn thuc l) - 3ssARN (+) H: Bromoviridae Ging: Ilavirus V d: Tobaco streak virus (virus sc ln thuc l) Cucumovirus V d: Cucumber mosaic virus (virus khm l da chut) Alfamovirus V d: Alfalfa mosaic virus (virus khm l c inh lng) + Cc virus c ARN si n m ( - ssARN): gm 90 loi

- 1(-) ssARN H: Rhadoviridae Nucleorhadovirus V d; Potato yellow dwarf virus (virus vng ln khoai ty) - 3 (-) ssARNs H: Bunyaviridae Ging: Tospovirus V d: Tomato spotted wilt virus (virus m ho c chua) - 4 (-) ssARNs Ging: TenuivirusV d: Rice stripe virus (virus sc nh l la) b) ARN si kp (ds ARN) + Virus hnh cu: 40 loi 2dsARN H: Reoviridae Ging: Fijivirus V d: Rice Fiji disease virus (virus bnh Fiji la) Oryzavirus V d: Rice ragged stunt virus (virus xon ngn l la) 7.2. Nhm ADN virus a) ADN si kp (ds ADN): 21 loi + ds ADN virus hnh cu: Ging Caulimovirus V d: Cauliflower mosaic virus (virus khm spl) + Virus hnh vi khun khng c v bc: Ging Badnavirus V d: Rice tungro baciliform virus (virus tungro dng vi khun hi la) b) ADN si n (ss ADN): 55 loi + Virus hnh chy Gy hi cy mt l mm, lan truyn qua ry H Geminiviridae Ging Geminivirus

V d: Maize streak virus (virus sc l ln ng) Gy hi cy hai l mm, lan truyn qua b phn. V d: Bean golden mosaic virus (virus khm vng cy u). + Virus hnh cu n V d: Banana bunchy top virus (virus chn ngn chui).

8. S TRUYN BNH VIRUS THC VT.

Virus thc vt xm nhp vo cy kho hoc truyn lan sang i sau ca cy trng bng nhiu con ng rt khc nhau hoc nh mi gii truyn bnh (vector) hoc khng nh mi gii truyn bnh. Nhng ni chung l virus khng th t lan truyn, chng lun phi nh mt s tr gip bn ngoi c th ly lan. Ta c th tm chia cch truyn bnh ca virus thc vt lm 2 nhm: 8.1. S truyn bnh virus khng nh mi gii. a) Truyn bnh qua nhn ging v tnh thc vt - Qua nui cy m: virus c th truyn d dng qua nui cy m t bo thc vt. Nu t bo b nhim virus c em nui cy v nhn ln s lng ln th nhng cy con c to thnh c th b nhim bnh t 90 - 100% mc bnh khc nhau. - Truyn qua hom ging chit t cy b bnh, qua mt ghp, cnh ghp, chi ghp, gc ghp b nhim bnh. Cc cy trng nhn ging v tnh bng c nh khoai ty, mt s cy hoa, cy cnh, bng c v thn nh khoai lang, sn... lun c nguy c b virus ph hoi trn din tch ln, nu khng kim sot c ngun ging ban u. b) Truyn bnh qua ht ging v qua phn hoa Virus thng khng truyn qua ht ging Bnh virus truyn qua phn hoa khng ch nhim vo ht ging m c th nhim vo cy con hay mm mc t ht ging . Quan trng hn chng c th truyn qua trong qu trnh th phn hoa xm nhp vo cy m. Virus cng c th qua phn hoa m ly t cy ny qua cy khc trn ng rung. C khong 100 virus lan

truyn c qua ht ging. Tuy nhin, trong thc t ch c mt s t ht mang virus. Mt vi trng hp c bit nh: TRSV t l nhim bnh rt cao c th n 100% s ht cy bnh (cy u tng). BSMV nhim ht cy la mch t 50 -100%, virus TMV nhim ngoi v ht (Barley). Ht cy h u b nhim nhiu loi virus (u tng v cc loi u n qu). Do cn ch chn lc ging sch bnh chng virus nhim ht to cy con b bnh thnh ngun bnh ban u nguy him cho cy trng ban u sau ny. c) Virus truyn bnh bng c hc, tip xc Truyn bnh virus bng c hc tip xc thng xy ra vi nhm cc bnh virus c tnh chng chu cao vi iu kin mi trng. Trong thin nhin khi cy mc dy, giao tn nhau bnh c th truyn khi l cy bnh c st vo l cy kho, c bit l cc rung trng rau v cc cy trng hng nm. Thng trong ma ma bo nc ta, khi gi mnh t cp 3, 4 tr ln d gy ra vt thng cy nn t l cy nhim bnh c th cao hn. Cc vt thng gy nn do cn trng, cc ng vt khc, my mc, dng c. Khi chm bn, thu hi to cc vt thng thn cy, l, r, cy kho l iu kin cho virus dng git dch ly nhim t cy bnh sang. Cc virus khm khoai ty X, virus bnh khm l cy thuc l l cc virus d dng ly bnh qua cc vt thng c gii, tip xc. Cc virus c kh nng chng chu km hn vi iu kin ngoi cnh nh virus Y, virus A khoai ty ly truyn c hc yu v truyn cn trng d dng hn. Virus c kh nng truyn bnh qua cc vt thng nh. 8.2. S truyn bnh virus bng mi gii Mi gii (vector) l cc vt trung gian gip cho virus c th t mt cy bnh xm nhp vo cy kho thc hin qu trnh xm nhim, gy bnh. T nm 1895 Takata, 1901 Takami (Nht Bn) pht hin b ry Inazuma dorsalis v b ry Nephotettix cineticeps l mi gii truyn bnh la ln cy Nht Bn. Nm 1916 Doolittle pht hin ra rp Aphis gossypii truyn bnh khm l da chut. Nm 1920, O.Botjes thy rp Myzus persicae truyn bnh cun l khoai ty.

Theo Harris (1981) c ti 381 loi ng vt c th truyn bnh virus hi thc vt, trong 94% thuc ngnh chn khp (athropoda) v 6% thuc ngnh giun trn (Nematoda). Cn trng l nhm mi gii c bit quan trng chim ti 99% cc loi thuc ngnh chn khp truyn bnh virus thc vt. a) Cc phng thc truyn qua mi gii Virus truyn bnh bng mi gii c th c nhiu kiu truyn bnh khc nhau: - Truyn bnh theo kiu truyn sinh hc ngha l c mi quan h sinh hc gia virus v c th cn trng, virus c mt thi gian tim n trong c th cn trng, qua tuyn nc bt i vo h thng tiu ho thm qua thnh rut vo mu ri li tr v tuyn nc bt. Sau giai on tim n ny virus c th c tng nng do c c c hay trong mt vi trng hp virus c th sinh sn ngay trong c th cn trng. Cc tc gi chia cc kiu truyn bnh qua cn trng v cc ng vt thnh 3 nhm virus. + Nhm truyn theo kiu bn vng: l nhng virus c th sng bn vng trong c th cn trng mt thi gian v t mt vi ting n mt vi tun l mi c kh nng ly bnh cho cy. V d: - virus gy bnh xon l c chua (Tomato leafcurl virus) - Virus gy bnh cun l khoai ty (Potato leafroll virus) + Nhm truyn bnh theo kiu khng bn vng. Gm nhng virus khng c kh nng tn ti trong c th cn trng t mt vi pht ti 1 gi. l nhng virus ly bnh nhanh chng trong khong thi gian t 15 giy n 30 pht chnh ht cy bnh sau c th ly lan ngay. in hnh l virus thuc nhm Potyvirus nh: - Bnh khm ln cy ng (Maize dwarf mosaic virus) - Bnh khm vng l u (Bean yellow mosaic virus) + Nhm truyn bnh na bn vng Nhng virus thuc nhm ny c kiu truyn bnh trung gian gia hai nhm trn. C th k in hnh l virus Tungro hi la, virus Tristeza hi cam chanh...

b) Cn trng truyn virus Cn trng chim ti 99% s loi thuc ngnh chn khp (athropoda, Harris, 1981), cc loi cn trng c th truyn bnh virus hi thc vt thuc cc b: - B cnh u (Homoptera) - B cnh na (Hemiptera) - B cnh cng (Coleoptera) - B cnh thng (orthoptera) - B cnh t (Thysanoptera) l nhng b c nhiu h v loi cn trng truyn bnh. Cc h rp mui (Aphididae), h ve su (Cicadellidae), h mui bay (Delphasidae), h ve su sng (Menbracidae), h b phn (Aleyrodidae), h rp gi (Pseudococidae). Cc b, h cn trng gm rt nhiu loi. Theo A.Gibbs v B. Harrison (1976) c khong 400 loi v c th truyn hn 200 virus khc nhau gy nhiu bnh hi cy trng. Ch ring rp o (Myzus persicae) thuc h rp mui c th truyn ti 60 bnh virus. Cc loi rp, b ry, b phn, v.v... phn ln u chch ht dch cha virus t b mch phloem ca cy, virus ctruyn c th thuc nhm bn vng, khng bn vng hay na bn vng tu thuc c tnh ca virus thuc nhm no v mi quan h gia chng vi cn trng. C loi rp c th truyn c 3 loi virus thuc 3 nhm, c loi ch truyn 1 virus thuc mt nhm, iu ny ph thuc vo mi quan h sinh hc gia cn trng v virus. C loi rp khi ht virus persistant n c th gi virus c i trong c th, song khi ht virus non - persistant n ch gi virus tuyn nc bt trong khong 15 giy n 30 pht nh rp o, b ry (Nephotettix apicalis) c th gi virus bnh la ln qua c th c i v c th truyn qua trng ti 7 i sau. Tui ca cn trng cng rt quan trng, ni chung cc cn trng t tui 3 - 5 c kh nng truyn bnh nhiu hn cc cn trng cn non.

c) Nhn truyn virus thc vt Nhn thuc loi tm chn, chng c mt kh cao trn cc cy k ch nhng phm vi k ch ca nhn hp hn cc loi cn trng khc. Theo kt qu nghin cu ca nhiu tc gi, nhn l mi gii truyn mt s loi thuc h eriophyidae, c kch thc khong 0,2mm, y l nhng loi nhn rt nh v c phm vi k ch hp. Loi nhn h Tetranyhidae c kch thc di 1mm c phm vi k ch kh rng. Loi Tetranychus telarius (Schultz, 1963) hay loi T. urticac (Koch) c th truyn virus PVY. Loi nhn Aceria tulipae truyn virus gy bnh khm sc l la m, chng c th chch ht trong 15 pht. Virus khng truyn qua trng nhn (Slykhui, 1955). d) Virus truyn bnh nh tuyn trng C hn 20 virus c truyn nh tuyn trng, cc ging Trichodorus, Paratrichodorus, Longidorus ging Xiphinema... Cc loi tuyn trng thng truyn nhng virus khng bn vng nh bnh ho nu sm u H Lan (Pea early browning), bnh gin l thuc l (Tabaco rattle virus) truyn bnh bng nhng tuyn trng trng thnh ca hai ging Trichodorus v Paratrichodorus. Mt s ging tuyn trng c th gi trong c th chng mt thi gian kh di, mt vi thng thm ch hng nm (Van Hoof, 1970). Cc nhm Tobraviruses, Nepoviruses l nhng nhm virus thng truyn bnh nh tuyn trng. e) Bnh virus truyn nh nm Mt s loi nm gy bnh cy, trong qu trnh gy bnh xm nhp vo cy kho c kh nng mang theo virus thc vt xm nhp v gy bnh cho cy, c bit l cc loi nm sng di t nh nm Olipidium, Polymyxa v Spongospora. Cc nm ny thng sinh bo t ng (zoospore) xm nhp vo r cy kho v gy bnh cho cy. Nm Olipidium thng truyn cc loi bnh virus: -Virus gy m cht hoi thuc l (Tabaco necrotic virus) -Virus gy m cht hoi da chut (Cucumber necrotic virus)

-Virus ci cc cy thuc l (Tabaco stunt virus) Nm Polymyxa truyn bnh khm l la m (Wheat mosaic virus) v bnh m cht vng gn cy c ci ng (Beet necrotic yellow vein virus). Nm Spongospora truyn bnh qut ngn khoai ty (Potato moptop virus). f) Virus truyn bnh bng dy t hng Qu trnh truyn bnh ny thng xy ra chm, n ph thuc vo s sinh trng v pht trin ca cy t hng. Trong trng hp cy t hng pht trin trn cy bnh nhanh v mc lan sang cy kho sm th bnh cng c th ly nhanh, ngc li, cy t hng pht trin chm trn cy bnh th vic truyn bnh s ko di. Thi gian ko di t 5 - 6 thng hoc lu hn. Bnh thng ly cay di, cy ly g, cy n qu.

9. PHNG TR BNH VIRUS HI CY TRNG.

Hin nay trn th gii ngi ta pht hin ra khong 650 loi virus hi thc vt (Yohashiro v ctv, 1991), trong c nhiu bnh hi c ngha kinh t. Bnh virus khng ch lm gim nng sut cy trng m cn l mt trong nhng nguyn nhn gy thoi ho ging cy trng. Theo Reesman A. J., 1970 chu u virus cun l lm gim nng sut 50%, virus Y v virus A lm gim nng sut 50% vi triu chng nng, virus X v virus S lm gim nng sut 25%. Bnh virus hi c chua lm gim nng sut t 15 - 25% (Broadbent, 1976), bnh virus hi thuc l lm gim thit hi 5,2 triu la trong nm 1978 ti nam Carolina (Gooding v Main, 1981). Bnh khm l cy ng cc gy thit hi c tnh t 3 - 14 triu USD (Sill v ctv, 1955) M. Bnh Tungro Philipines nm 1971 gy thit hi trn na triu tn thc,v.v. i vi cy lu nm nh cam, chanh, mn, l, to bnh virus khng nhng lm mt hoc gim nng sut, cht lng qu m cn l ngun bnh nguy him cho nhng nm sau. Virus c th truyn qua tip xc c hc, qua ht ging, hom ging, nui cy m, cn trng mi gii, nm, tuyn trng, thc vt thng ng k sinh,... Do tnh cht gy hi ch yu trong h mch dn, kh nng pht tn nhanh chng qua con ng trao i ging v s truyn lan ca cn trng mi gii nn bnh c mc pht trin mnh, d gy thnh dch. y l mt trong nhng loi bnh kh phng tr, cc bin php ho hc t c tc dng. 9.1. Cc bin php phng tr bnh virus hi thc vt

Trn th gii nhiu bin php phng tr bnh virus hi thc vt c p dng nh loi b ngun bnh, tiu dit cn trng mi gii, dit c di, lun canh cy trng, dng ging sch bnh hoc ging chng bnh, chu bnh. Da vo c im ca tng loi virus gy hi, c tnh cy trng ngi ta ra nhng bin php phng tr cho tng nhm bnh theo kh nng truyn lan v s tn ti ca ngun bnh. * S dng ht ging, cy trng sch bnh Mt loi virus c kh nng truyn qua ht ging v d: - Virus khm l u tng (SMV) (Jame v ctv, 1982) - Khm m cy lc (PMV) (Jame v ctv, 1982) - Virus khm m xanh l da chut (CGMV) (Holling v ctv, 1975) - Virus khm l thuc l (TMV) (Tsuzuki v ctv, 1967; Nagai, 1981) Bin php: - Chn ht ging t cy kho, sch bnh (Jame v ctv, 1982; Y.Honda v ctv, 1977). - X l ht ging l bin php tch cc loi tr v phng bnh ly lan trn vn m: + X l nhit (ht t, c chua, da) x l khng kh nng 700C trong 2 - 3 ngy, x l nhit khoai ty ging 360 C trong 40 ngy c th hn ch c virus cun l (Duriat, 1989). + X l ht ging bng ho cht nh Trisodium photphat 10% hay c th dng Monazon hn ch bnh lan truyn qua tip xc c hc (Yokashi v ctv, 1991, Yohachiro v ctv, 1991). - Bin php nui cy m t nh sinh trng cng x l nhit, bin php ny c ng dng rng ri trong v ngoi nc nhn ging cc loi cy nh khoai ty (Kasanis, 1957; Pett, 1974; Nozeran, 1977). Qu trnh x l nhit nhit 32 380 C trong thi gian 7 ngy n 7 tun sau nui cy nh sinh trng c th loi tr cvirus A, xon l, virus X v virus Y trong khi virus M v virus S

cng gim i ng k. i vi cc loi rau c vt liu sch ban u ngi ta phi dng bin php nui cy m, sau em nhn ging v tnh vi s lng ln dng sn xut ht (Walley v ctv, 1974). Cy n qu thng l cy di ngy lun chu tc ng ca cc tc nhn truyn bnh, thi gian bnh thng ko di, gy kh khn cho vic lm sch virus. Virus nui cy nh sinh trng v x l nhit i vi cy thn g kh khn hn nhiu so vi cy thn c do kh nng ti sinh chi yu. Qu trnh x l nhit thng ctin hnh ch yu nhng giai on cnh mang mt ghp sau ny. Theo Nyland v Gohhen (1969) x l nhit c th loi tr c 6 loi virus cy anh o, 2 virus cy phc bn t, 2 loi virus cy nho, 7 loi virus cy to. Thnh cng trong x l virus cy n qu kh t 100%. Nui cy nh sinh trng cy n qu nay mi ch c s dng trn nhng i tng sau: to (Walley, 1972), du (Putz, 1974) v nc ta cng nh mt s nc trn th gii nui cy m cy chui, du ty c s dng rng ri trong vic to ngun ging sch bnh cho sn xut. Trn i tng l cy hoa, k thut nui cy m nhn ging v tnh c ng dng ti nhiu nc trn th gii. nc ta vi mt s loi hoa qu nh phong lan, thu tin, cc cng s dng phng php ny,.. H Lan, Anh, c vic sn xut cy sch bnh l vic lm thng xuyn v c cc c s chuyn nhn v lm sch virus trn cy hoa. Bin php nui cy m to ngun ging sch bnh kt hp chn lc v sinh ng rung trn cy khoai ty c nghin cu v ng dng Vit Nam (Nguyn Quang Thch, Hunh Minh Tn, V Triu Mn v ctv, 1990 - 1992). Nng sut khoai ty trong h thng chn lc v sinh ng rung tng t 2,4 - 4 tn/ha trn din tch rng (V Triu Mn, 1986). 9.2. Chn on v phng tr bnh virus hi thc vt Phng php chn on virus thc vt rt ph bin hin nay l chn on bng triu chng, bng khng huyt thanh v kit ELISA, bng phng ph PCR, phng php cy ch th v bng knh hin vi in t 9.2.1. Mc ch Chn on bnh cy nhm xc nh nguyn nhn gy bnh v cc biu hin bn ngoi ca bnh, phn bit r vi cc hin tng bnh do k sinh khc v do mi trng gy nn, t c bin php phng tr ng n. 9.2.2. Cc iu kin cn thit chn on bnh cy

a) Ngi lm cng tc chn on: chn on c bnh cy ngi lm cng tc chn on phi l ngi c o to chnh quy mn bnh cy v t nht c 3-5 nm tham gia cc hot ng iu tra, nghin cu bnh cy. b) Thng tin v cy v khu vc cn chn on: phi bit r cht t, ch chm sc, c im ging cy, giai on sinh trng, iu kin kh hu thi tit, ma v, cc bin php phng tr thc hin, cc cy trng v trc... c) Cn c nhng trang thit b v ti liu ti thiu chn on bnh chnh xc nh: knh hin vi quang hc, cc trang thit b khc nui cy vi sinh vt. Ti thiu c Kit ELISA xc nh (nu l bnh virus) c cc ho cht cn thit gip cho chn on nhanh v chnh xc. 9.2.3. Khi qut v cc bc chn on bnh cy Bc 1: Quan st bao qut ng rung nh gi mc ph bin ca bnh v ging b hi ch yu, mc hi v thi gian xut hin bnh. Bc 2: Phn bit triu chng bnh c bit khc vi cc bnh do k sinh khc v mi trng gy ra. Tm ra c nhng im c th ca b phn b hi. Bc 3: Xc nh c vi sinh vt gy bnh v c im ca chng i n kh nng phng tr c hiu qu v kinh t nht. Chn on bnh kh phc tp, l do ch yu l v cy bnh buc phi tn ti v pht trin trong iu kin sinh thi mi trng lun bin ng. Tnh trng bnh l li ph thuc loi, ging, tui cy v bn cht vi sinh vt gy bnh. Do , cn c tc phong linh hot v c bit khng b qua cc chi tit c bit thu c hiu qu cao. 9.2.4. Cc phng php chn on bnh cy a. Phng php chn on bng triu chng bn ngoi D chn on bng phng php no i na, th cui cng kt lun v triu chng bn ngoi vn l mt phng php rt quan trng trong chn on bnh cy. Thng qua cc biu hin bng triu chng bn ngoi, chng ta c th hiu bit t nhiu v nguyn nhn gy bnh bn trong v ngc li. iu quan trng nht trong chn on triu chng l phi tm ra c im ring bit ca tng loi nhm bnh

v tng loi nguyn nhn gy bnh c th so snh chng vi nhau, trnh mc phi nhng nhm ln. Lun lun phi lu mt hin tng: mt nguyn nhn gy bnh c th gy ra nhiu dng triu chng khc nhau v ngc li mt triu chng c th do nhiu nguyn nhn khc nhau gy ra. Triu chng bnh cn ph thuc vo mc nng nh khi bnh gy ra trn mt cy ph thuc vo ging cy khc nhau, chm sc khc nhau v iu kin sinh thi v kh hu khc nhau vo bn cht ca nguyn nhn gy bnh khc nhau c bit l tnh c ca vi sinh vt khc nhau. Chn on bng triu chng lun rt quan trng, rt kinh t v mang li hiu qu cao. Tuy nhin, trong chn on bnh cy nu ch s dng mt phng php c th cn phin din nn ngi ta thng dng nhiu phng php phi hp nhau kt lun nguyn nhn gy bnh mt cch chnh xc. b. Phng php chn on bng knh hin vi quang hc thng thng Nhng vi sinh vt c th kim tra bng knh hin vi bao gm nm, x khun, vi khunVirus, phytoplasma, viroide khng th s dng knh hin vi thng m phi dng knh hin vi in t phng i hng vn n hng chc vn ln quan st v chng rt nh b. Mun chn on vi sinh vt bng knh hin vi thng phi c mt s iu kin sau: - Phi nm vng phng php s dng knh hin vi quang hc - Thu mu nm, vi khun ngoi ng phi l mu c vt bnh ang pht trin hoc mi hnh thnh. Nu ly vt bnh c d nhm nguyn nhn gy bnh vi cc vi sinh vt hoi sinh, ph sinh ri ngu nhin v mc tp trn b mt vt bnh. - Nu vt bnh mi cha c bo t hay dch bo t th cn mu l bnh (thn, cnh, qu) vo hp m petri c lt giy m trong iu kin nhit phng hay trong t m nhit m (300C) hng ngy pht hin si nm v bo t xut hin trn b mt vt bnh ly mu quan st. - C th quan st trc tip bo t trn vt bnh di knh hin vi: v hnh dng, mu sc, o kch thc ca bo t, hoc dng phng php nhum methylen xanh, nitrat bc 10% t 3-5 pht, thm kh nh ri nhum tip vo dung dch KOH 10%, hay nhum KMnO4 5%, hoc Fucsin Fenol pht hin th si nm hay vi khun c trong m bnh.

- Khi quan st vi khun c th thc hin cc k thut chn on nhanh nh ngm 1 u l bnh vo dung dch NaCl 1% trong 15 - 30 pht v quan st git dch vi khun xut hin u l nh ln mt nc. Nhum gram, nhum lng roi, xem trn knh du phng i hn 400 ln v m t hnh dng, mu sc, o m kch thc, vi khun mt cch chnh xc. T bo vi khun cn c th c quan st r trn knh hin vi hunh quang khi dng phng php nhum khng th hunh quang vi khun. c. Phng php chn on sinh hc Vi vi sinh vt ch yu l nm v vi khun khi cn phi phn lp trn mi trng c th dng mt mu nh m cy mi nhim bnh. Ct phn l gn vt bnh cy vo mi trng, dng phng php pha long v cy truyn phn ly. Cc loi mi trng thng dng l: mi trng Water Agar (WA) (thng dng 20g Agar v 1000ml nc ct). Sau l cc mi trng phn lp nm (mPDA, CLA, PDA, CMA) mi trng phn lp vi khun (SPA, Kings B, TZC, Wakimoto, PS, PG, PGA). Trong cc mi trng, c nhng mi trng gi l mi trng tng hp (tt c cc cht u bit r thnh phn ho hc, thng l cc mi trng lng). Mi trng bn tng hp l mi trng c mt s cht hoc mt cht khng r thnh phn ho hc. V d: mi trng PGA: Khoai ty : 200g - cha r thnh phn ho hc Glucose : 20g Agar : 15g - cha r thnh phn ho hc Nc ct : 1000ml C mi trng gi l mi trng thin nhin (khng bit thnh phn ho hc ca cht to mi trng). V d : mi trng c khoai ty, mi trng c c rt, mi trng khoai ty Agar. v tnh cht vt l. Mi trng cn c th chia thnh dng mi trng lng v mi trng c (khi dng Agar). Nui cy vi sinh vt trn mi trng lng do thiu oxy thng phi dng my lc tng lng oxy cho mi trng. d. Phng php dng khng huyt thanh chn on bnh

Khng huyt thanh chn on bnh hi c th nghim da trn hin tng khi c mt cht l (khng nguyn) vo c th, c th s c kh nng khng li bng cch to p ng min dch hnh thnh khng th. Lc u, phng php ny s dng cho bnh virus nhng nay ph bin c trong chn on vi khun v mt s bnh khc. Khng th a dng (Polyclonal antibody) Khi ta tim dch virus c lm tinh khit t cy ch th b nhim bnh vo c th ng vt, c th ng vt s thc hin p ng min dch. Trong trng hp ny c th ng vt to nn nhiu khng th do nhiu dng t bo B to ra. chnh l polyclonal antibody hay gi l khng th a dng. Trong chn on bnh cy khng th a dng c hiu qu rt cao trong vic loi tr cy b bnh d chng chng no thuc cng mt loi vi sinh vt gy bnh. Khng th n dng (Monoclonal antibody) L khng th khng nhn ln trong c th ng vt m nhn ln trong t bo ung th c nui cy trn1 bn plastic. Tm tt phng php to khng th n dng ca Milstein v Kohler (1975) nh sau: T bo lympho B c gen m ho Ig (to khng th) + T bo u tu Myecoma (nhn nhanh) ca mt ng vt b ung th. Hai t bo ny dung hp vi nhau v c nui trong mi trng HAT (cha hypoxantin, aminorperin v tomidin) chng to ra t bo lai. Thc hin nui cy n bo trn bn plastic trong iu kin v trng tuyt i ta thu c dng 1, 2, 3, 4,T sn xut c khng th n dng (monoclonal antibody). Khng th n dng c th pht hin ti chng (strain) ca virus hay ni (race) ca vi khun v nm gy bnh hi thc vt. Khng nguyn (virus c trong dch l bnh) s kt hp vi khng th (c trong khng huyt thanh) u to kt ta d l khng th n dng hay a dng. Khng huyt thanh lun c tnh c hiu cao: - khng nguyn A ch kt hp vi khng th A. - khng nguyn B ch kt hp vi khng th B. - khng nguyn C ch kt hp vi khng th C.

Khng c hin tng kt ta cho gia A, B, C, chnh v vy chng ta c th s dng khng huyt thanh chn on xc nh bnh hi. Trong sut nhng nm u ca th k 20 (1930 - 1970) khng huyt thanh l phng php chn on rt quan trng v phn ng xy ra nhanh t 15 - 20 pht trong iu kin nhit phng th nghim khong 20 - 250C. Nhng quan st phn ng ngy mt kh hn khi ta gp cc trng hp kt ta qu t kh c th phn on c hay khng c phn ng (phn ng ). Nm 1977, Clark v Adam (Scottlen) dng phng php th nghim min dch lin kt men (Enzyme linked immunosorbent assay ELISA) ln u tin trn thc vt. Phng php ELISA to ra mt s i mi trong vic s dng khng huyt thanh lm tNng chnh xc ln hng nghn ln. Bn cht ca phng php vn l s dng khng nguyn v khng th to ra s kt hp gia chng vi men (enzyme) lin kt - nhng ch th ca phn ng khng phi l kt ta m l mu vng. My c ELISA khc phc hin tng mu vng nht dn v cung cp cho chng ta bng s liu ch r cc phn ng xy ra tng ging trong bn ELISA. Phng php ELISA direct (DAS - ELISA), phng php indirect l nhng phng php s dng ph bin trn th gii cho n nay, nhng phng php ny vn dng rng ri trong sn xut v c coi l phng php huyt thanh chnh xC nht c s dng hin nay. Chi tit cc quy trnh ca phng php ELISA nh sau: Phng php DAS - ELISA (Double antibody sandwich - ELISA) hay cn gi l phng php ELISA trc tip Bc 1: C nh IgG c hiu ca virus vo bn ELISA. IgG ho trong dung dch m carbonate, cho vo mi ging 100 ml. t bn ELISA trong hp m, vo t m nhit 370C trong thi gian 2 - 4h. Sau khi , ging c ra bng dung dch m ra (PBS - T) ba ln, mi ln trong 3 pht. Bc 2: C nh dch cy vo bn ELISA. Nghin mi mu 2 g trong m chit (PBS - T + 2% PVP) vi pha long 1/10 v 1/20. Dch cy c nh vo bn ELISA vi lng 100ml /ging. Sau t bn ELISA vo hp m trong t lnh - 40C qua mt m hoc c th 370C trong khong 2 - 4h. Trong qu trnh ny IgG s xy ra lin kt gia IgG v

khng nguyn (nu mu l mu b nhim bnh). Sau khi bn ELISA c ra nh bc 1. Bc 3: C nh IgG lin kt enzyme. Ho IgG lin kt enzyme (IgG - E) trong dung dch m lin kt (PBS -T + 2% PVP + 0,2% Ovalbumin) theo t l cho vo ging vi lng 100 ml/ging. Bn ELISA c 370C trong 2h v ra nh bc 1. Bc 4: C nh cht nn vo bn ELISA. Ho cht nn NPP (nitrophenol phosphate) vo dung dch m substrate (theo t l 0,25 - 0,5mg/1ml dung dch m). Sau nh vo mi ging 100 ml. Bn ELISA trong hp m c t nhit trong phng th nghim. Sau 1h cc ging c mu vng l ging c phn ng dng tnh, ging khng mu l khng c phn ng. Kt qu c c chnh xc hn trn my c ELISA (ELISA reader) bc sng 405 nm. c nh mu sc ca bn ELISA, bo qun trong t lnh 40C nu cn xem li vo khi khc c th dng dung dch NaOH 3M nh vo mi ging 25 - 30 ml. Phng php Indirect ELISA hay cn gi l phng php ELISA gin tip Bc 1: C nh dch cy (nghi l b bnh) cn kim tra vo bn: cn mi mu 0,2 g l cho vo ti nilon nghin trong dung dch PBS vi t l l/dung dch m l 1/20 - 1/100, nh vo bn 100 ml /ging. Sau bn ELISA vo hp m v qua mt m nhit 40C. Bc 2: Chun b mu cy kho (cy c kim tra ELISA khng b nhim) nghin trong dung dch m pha huyt thanh (PBS - T 1000ml + 2% PVP + 0,2% Ovabumin) theo t l 1/20. Lc qua vi lc ta thu c dch cy kho. Cho khng huyt thanh vo dch cy kho theo nng pha long tu tng loi khng huyt thanh khuy u v 45 pht trong iu kin 370C. Bc 3: Ra bn ELISA vi m PBS - T 3 ln trong 3 pht.

Bc 4: C nh khng huyt thanh vo bn ELISA, nh vo mi ging 100 ml khng huyt thanh pha long trong dch cy kho. Sau cho bn ELISA vo trong hp m v iu kin nhit 370C trong thi gian t 1 - 1h 30 pht. Bc 5: Ra bn ELISA nh bc 3. Bc 6: C nh khng huyt thanh ca khng nguyn IgG th (hay khng huyt thanh B (conjugate AP) vi ho long 1/1000 - 1/2000. Mi ging 100 ml. t bn ELISA vo hp v qua mt m t lnh 40C (hoc nhit 370C trong 1h - 1h 30 pht). Bc 7: Ra bn ELISA nh bc 3. Bc 8: C nh cht nn v nh gi kt qu: - Pha 0,25 - 0,3 mg NNP/1ml m subtrate ri ho tan bng my khuy t. - Sau nh dung dch trn vo bn ELISA, 100 ml/ ging. a bn ELISA vo hp m v trong nhit phng th nghim (khong 200C) trong thi gian t 30 - 60 pht. Bc 9: c kt qu: Cc ging c mu vng l cc ging c phn ng (+). Ging khng c mu l cy khng b nhim bnh. c kt qu tip bng cch a vo my c ELISA bc sng 405 nm. Cng c th dng phn ng bng NaOH 3M vi lng 25 - 50 ml/ging nh phng php DAS - ELISA. e. Cc phng php chn on sinh hc phn t Phng php chn on khng huyt thanh v ELISA l nhng phng php chn on protein. Cho ti nay (2006) vn l phng php c ng dng rng ri chn on virus ngi, ng vt v thc vt ng cc hng Agdia, Biorad (M), nhiu hng sn xut ca Nht, c, Php, H Lan...thng mi ho v a ra th trng rt nhiu sn phm do gi tr ca cc sn phm ny r v chnh xc cao. Nhng phng php chn on sinh hc phn t l phng php pht hin ARN v ADN. T nhng nm 80 khi phng php sinh hc phn t ra i th vic xc nh virus thc vt bt u pht trin mc phn t. C rt nhiu phng php sinh hc phn t c ng dng, song ti nay PCR (polymeraza chain

reaction) l phng php c s dng rng ri v mang li hiu qu cao nht. Phng php c thc hin trn c s kh nng ti t hp ca ADN, ARN invitro. Mun thc hin kh nng ny cn c cc iu kin c bn sau: Tch c 1 lng nh ADN nguyn bn, trn vi mt tp hp cc cht trong mi trng mui m gm Taq Polymeraza, dNTPs (Deoxyribonucletit triphophates), MgCl2. Mt hoc hai on nucleotit lm mi (primer). Tm tt cc bc ca phng php PCR gm: - Bc 1: Si ADN kp c x l 940C trong 5 pht to thnh 2 si n. - Bc 2: on b sung si n ADN v on mi ghp cp 30 - 650C trong 3 giy. - Bc 3: Tng hp si n mi ADN 65 - 750C trong 2 - 5 pht. - Bc 4: Quay tr li bc 2 sau khi ADN kp li tch thnh 2 si n 940C trong 30 giy.sn phm PCR c in di trn gel Agarose hoc gel Polyacrylamide. g. Phng php hin vi in t Phng php knh hin vi in t l phng php quan trng pht hin cc virus, phytoplasma, viroide gy bnh thc vt m knh hin vi thng thng vi phng i nh khng thc hin c. Max Knol, Ernett Ruska (1931) l nhm cc nh khoa hc ch to thnh cng knh hin vi in t. T , virus thc vt v ng vt c quan st chnh xc v hnh thi, cu to. Hin vi in t c hai loi knh ch yu l: Knh hin vi in t truyn qua (transmission electron microscope) v hin vi in t qut (scaning electron microscope). Knh hin vi in t truyn qua l loi knh c s dng ph bin trong nghin cu bnh cy. thc hin c k thut quan st v chp nh hin vi in t c th xem trc tip mu (phng php DIP) hoc xem mu vi sinh vt (ch yu l virus) c lm tinh khit. Hoc ct lt ct m bnh cc mng bng my ct siu mng (Ultra microtom) quan st virus trong m t bo cy b nhim bnh. Tt c cc phng php to mu trn u s dng nhiu thuc nhum v ho cht c nh mu vt v khi thc hin lt ct siu mng phi tin hnh vi my ct trong chn khng. T phng php hin vi

in t lm c s ngy nay ra i v pht trin nhiu k thut mi nh hin vi lc nguyn t, hin vi t lc, hin vi qut hiu ng ng ngm. h. Cc phng php khc i vi mt s bnh hi thc vt trc y ngi ta dng mt s phng php n gin vi chnh xc khong 80% chn on s b bnh hi: - Dung dch Rezocin 10% khi nhum mu lt ct mng c khoai ty pht hin thy cc b mch libe b nhum mu xm l hin tng c b nhim virus cun l (Potato leafroll virus - PLRV). - Dung dch sunfat ng CuSO4.5 H2O 3% nhum mu nu khi x l m cy h c, h bu b c th pht hin s nhim bnh do virus Cucumber mosaic (CMV). - Phng php gim nh nhanh bnh vng l Greening bng nhum Iod v dng giy th NCM cho kt qu tt. - Phng php o nht, c ca dch cy cng l phng php chn on s b bnh hi trong trng hp cy b bnh dch cy thng c c cao hn. - Phng php hunh quang chn on m qu, ht b bnh da vo s pht sng ca m bnh khi ta chim ngun sng t n thch anh c bc sng khc nhau... - Phng php chn on bnh trn ht ging trc khi gieo trng: Sau khi ly mu kim tra ht theo quy nh, c th thc hin phng php ra ht ri ly tm nh v quan st dch thu c trn knh hin vi pht hin bo t hay si nm bnh hi. Dng cch kh trng t th nghim ri ngm ht v gieo trn t v trng pht hin cy con b bnh. Dng phng php ELISA v PCR chn on bnh c hay ht ging. - Vi tuyn trng: ngi ta ly mu t chiu su t 5 - 20 cm v s dng phng php Bekman (1995). Lc tuyn trng qua li lc 25 mm sau khi ngm mu t t 24 - 48 h. Hoc ngm r cy trong cc nc 2 - 3 h ta thu c tuyn trng y cc v dng knh lp phng i 50 ln quan st.

- Phng php pht hin git dch vi khun: c th dng l (bnh bc l la) hay thn (bnh ho xanh cy h c) ngm vo dung dch 1% NaCl hoc nc sch, sau 20 - 30 pht s thy git dch vi khun xut hin u l hay lt ct thn nh ln mt nc.