Nghiên Cứu Hệ Nấm Cộng Sinh Arbuscular Mycorrhiza, Trong Đất Và Rễ Cam Tại Quỳ...

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I HC THI NGUYN

TRNG I HC KHOA HC -------*-------

L TH THY

NGHIN CU H NM

CNG SINH ARBUSCULAR MYCORRHIZA,

TRONG T V R CAM TI QU HP - NGH AN

LUN VN THC S CNG NGH SINH HC

Thi Nguyn - 2012


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M U

S cng sinh gia nm v r cy trng c pht hin ln u tin vo

nm 1885 do A.B.Fank nh bnh cy lm nghip ngi c. Nhng phi

n nm 80 ca th k 20 mi c tp trung nghin cu v ng dng trong

sn xut.

Nm r cng sinh l hin tng rt ph bin trong t nhin, c khong

60 80% cc loi thc vt trn th gii c mi quan h cng sinh vi nm ni

cng sinh. y l mi quan h cng sinh khng th tch ri: Nm khng c r

th khng th tn ti, cy khng c nm cy sinh trng yu vng v cht

[11]. c nhiu cng trnh khoa hc chng minh vai tr ca nm cng sinh

mang li nhng li ch to ln, thit thc i vi qu trnh sinh trng v pht

trin ca cy trong iu kin bt li ca mi trng, bi vy ch trong iu

kin t ai kh hn, ngho dinh dng th nm r mi pht huy tt vai tr

cng sinh ca mnh. Chnh v vy, hnh thc cng sinh ny v ang c

nghin cu (v phn loi, sinh hc phn t, nh hng ca chng i vi thc

vt...) v ng dng vo thc tin sn xut nng lm nghip nhiu nc

trn th gii.

Vit Nam, din tch t trng trn cn l rt ln (3 317 270 ha), ch

yu l cc loi cy trng c gi tr kinh t cao. Tuy nhin, trong pht trin sn

xut thng gp kh khn v vn nc ti, t chua v dinh dng . V

vy, vic nghin cu p dng k thut pht trin nm cng sinh Mycorrhiza

cho mt s cy trng chnh ti cc vng sinh thi phc v sn xut Nng

Lm nghip bn vng nc ta nhm nng cao nng sut cy trng, duy tr

v bo v v nng cao ph nhiu ca t l vn cp bch cn c quan

tm hin nay. Cho n nay, mt s nh khoa hc ca Vin Lm nghip, Vin

Cng ngh sinh hc, Vin Th nhng nng ha cng cng b nhng

nghin cu c bn v nm r cng sinh. Kt qu ca nhng nghin cu ny


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mi ch dng li mc phn lp, lu gi bo t nm cng sinh v nghin cu

x ly t nhim chi co s dung nm Mycorrhiza. Tuy nhin vn cha c

nghin cu v kh nng cng sinh ca nm cng sinh trn cy cam nhm tao

ra ch phm lam tng nng sut va cht lng.

Trong cc loi cy n qu, Cam Vinh l mt loi cy n qu c sn

truyn thng c gi tr dinh dng cao, ng thi cng rt c gi tr v kinh

t. Tuy nhin, din tch trng cam y ang dn b thu hp v cht lng

ca cam ang ngy cng b mai mt. Vi mong mun c th gp phn no

vo vic ci thin nng sut, cht lng cam, nhm nghin cu tin hnh

nghin cu ti: NGHIN CU H NM CNG SINH ARBUSCULAR

MYCORRHIZA, TRONG T V R CAM TI QU HP - NGH AN. Vi mc tiu

v ni dung nghin cu sau:

Mc tiu nghin cu :

Nghin cu h nm ni cng sinh (Arbuscular Mycorrhiza Fungi) trn

t trng cam X Minh Tn - Ph Qy Ngh An. Trn c s , gp phn

xut cc gii php v phn bn, canh tc nhm lm tng nng sut, cht

lng ch, n nh ph nhiu, ci thin mi trng t vng trng cam.

Ni dung nghin cu:

Xc nh thnh phn loi AMF ti vng nghin cu.

Xc nh c im phn b AMF trong t trng cam ti Ph Qy

Ngh An.

Xc nh loi AMF trong t trng cam bng k thut sinh hc phn

t.


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CHNG I

TNG QUAN TI LIU

1.1. LCH S HNH THNH TN GI ARBUSCULAR MYCORRHIZA

FUNGI (AMF)

Thut ng Mycorrhiza ln u tin c Frank nh bnh cy lm

nghip ngi c a ra vo nm 1885 ch mi quan h c bit gia r

cy v nm ngoi cng sinh [53]. S cng sinh cua Mycorrhiza vi r c

m ta nh (hnh 1.1). Thut ng ny bt ngun t ch Hy Lp: Mykes (nm)

v Rhiza (r). Nm 1887, Frank ch ra s khc bit gia nm ngoi cng

sinh v nm ni cng sinh, thc cht l s khc bit gia Ericaceous v

Orchid, tng c gi l Phycomycetous Endomycorrihiza phn bit vi

dng cng sinh ca nm bc cao vi cc loi trong h Ericaceae v

Orchidaceae. Tuy nhin, tn gi ny tn ti khng lu v khng c ngha

[33]. Nhng nghin cu tip theo v cu trc dn n s thay i tn gi

ca hnh thc cng sinh ny. Nm 1897, tc gi Janse gi cu trc dng

bng bn trong t bo r ca thc vt b nhim nm Mycorrhiza l

Vesicules (gi l th V). Nm 1905, Gallaud gi nhng cu trc dng bi

(chm) trong t bo thng c quan st thy l Arbuscular (gi l th A).

Do vy, tn gi Vesicular Arbuscular Mycorrhiza (vit tt l VAM) c

hnh thnh v tn ti cho n thi gian gn y [19]. Bn cnh , mt s bi

bo v cng trnh khoa hc khc cn s dng tn gi Vesicular A

rbuscular Mycorrhiza Fungi ch loi hnh cng sinh ny [23]. Nhng

nghin cu sau ny cho thy, th A l c im chung nht ca cc chi nhng

khng phi tt c nm ni cng sinh u hnh thnh th V. Do vy, loi hnh


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cng sinh ny c th c i tn l Arbuscular Mycorrhiza. Ni chung,

tn gi ca n vn cha hon ton thng nht.

Nm 2005, Hi ngh Quc t v Mycorrhiza ln th 17 c t chc ti

Lisboa B o Nha quyt nh ly tn Arbuscular Mycorrhiza Fungi

(AMF) ch loi hnh cng sinh ny. Do vy, trong cc ti liu mi c

cng b, thut ng Arbuscular Mycorrhiza Fungi (vit tt l AMF) c

thng nht s dng thay cho thut ng Vesicular Arbuscular Mycorrhiza

vo nm 2008 [28].

Hnh 1.1. S cng sinh ca AMF trong r cy trng.

1.2. PHN LOI MYCORRHIZA

c im nhn dng ca mt s loi bo t AMF (da theo phn loi

ca Gerdemann, 1963) [32] bng 1.1.

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Bng 1.1. c im nhn dng ca mt s loi bo t AMF

Tn chi Tn loi Hnh dng Mu sc Kch

thc (m)

c im

Glomus

Aggregatum Hnh cu, hnh trng, c cung nh C mu nu, mu nu , mt s c

mu vng nht 120-175 Thnh bo t mng, c 2 lp.

Ambisporum Hnh cu hoc hnh trng Mu nu, nu m 100-150 Thnh bo t mng.

Marcaropus Hnh cu hoc hnh trng Nu hoc mu vng nht 100-125 Thnh bo t mng, c qu bo t.

Acaulospora

Appendicul

a Hnh cu C mu vng nht 150-175 Thnh bo t dy, 1-6 m.

Delicate c qu bo t, hnh cu C mu nu, nu m 100-175 Thnh bo t dy.

Dilatata Hnh cu, c qu bo t C mu nu, en 125-200 Thnh bo t dy,c nhiu lp.

Myriocarpa, C qu bo t, hnh cu hoc gn

hnh cu. C mu nu hoc nu nht 120-175 Thnh bo t dy, c vch ngn.

Bireticulata Hnh cu, dng qu l C mu nu nht hoc vng nht 150-200 Thnh bo t dy, c 3-4 lp.

Lacunose.

Hnh cu C mu nu hoc nu nht 120-175 Thnh bo t mng,1-2 lp

Entrophora

colombiana

Hnh cu hoc hnh trng C mu nu nht hoc mu vng nht 120-200

Thnh bo t c 3 lp chia lm 2 nhm

schenckii.

Hnh cu ,hnh trn C mu nu nht hoc mu vng 150-200 Thnh dy chia bo t thnh 3 lp.

Sclerocystis

Coccogena

Bo t c dng hnh cu, gn hnh cu, elip

C mu nu nht 100-150 Thnh bo t mng c 2 lp.

Coremioides Bo t c hnh cu, C mu nu, nu m 120-180 Thnh bo t c 3 lp,chia lm 2

nhm.

Glomites Rhyniensis Hnh cu hoc hnh elip C mu nu m 150-200 Thnh bo t c cu trc kiu lin

tip, gm nhiu lp.

Gigaspora

Candida, Bo t thng c hnh cu v gn

hnh cu, mt s c hnh elip C mu nu, nu m, mu nu 120160

Thnh bo t c 3 lp chia lm 2 nhm

Albida Bo t c dng hnh cu, hoc elip C mu vng nht 120-150 Thnh bo t c cu trc ni tip,

gm 3 5 lp.

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Cn c v mt hnh thi c th chia nm r thnh 3 loi ch yu: Nm r

ngoi cng sinh, nm r ni cng sinh v nm r ni ngoi cng sinh.

* Nm r ngoi cng sinh (Ectomycorrhiza): y l mt loi nm

hnh thnh mng li Hartig trong gian bo tng v r v m si nm dy

c trn b mt r ca cy, khng c m r, khng c lng ht. Nm r

ngoi cng sinh thng c mu sc v hnh dng nht nh (c th nhn

thy bng mt thng).

* Nm r ni cng sinh (Endomycorrhiza): c trng l khng c s

bin i mu sc v hnh thi ca r, c lng ht, khng c th si nm v

khng c mng li Hartig. Nm r ni cng sinh gm 2 loi l: nm r ni

cng sinh khng c mng ngn (AEM) v si nm ni cng sinh c mng

ngn (SEM). Vi loi SEM, khi gii phu s thy bn trong t bo biu b r

c cc ti bt (Vesicular) v chm (Arbuscular).

* Nm r ni ngoi cng sinh (Ectendomycorrhiza): Mang c trng

ca 2 loi ni cng sinh v ngoi cng sinh v hnh thi cng nh sinh l.

Hin nay, ngi ta nhn thy c 7 hnh thc cng sinh:

* Arbuscular Mycorrhiza Fungi (AMF): Cng sinh kiu to bi (chm).

* Ectomycorrhizas (ECM): Nm r ngoi cng sinh.

* Orchid Mycorrhizas: Nm r cng sinh vi cc cy h Lan

(Orchidaceae).

* Ericoid Mycorrhizas: Nm r cng sinh vi cc cy thuc b

Quyn (Ericales).

* Ectendo Mycorrhizas: Nm r ni ngoi cng sinh.

* Arbutoid Mycorrhizas: Nm r c c ni cng sinh v ngoi cng sinh

nhng ch xut hin gii hn trong cc chi Arbutus, Arctostaphylos v Arctous

ca h Quyn (Ericaceae).

* Monotropoid Mycorrhizas: Nm r cng sinh xut hin trong h

Monotropaceae ca b Quyn (Ericales).

Theo thng k ca Harley (1959), khong 3% s cy c hoa cc loi cy g

v cy bi c ECM, 90% cc loi cy thn c c AMF, ngoi ra mt s loi

cy g c c ECM v AMF [32].

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Hnh 1.2. Biu cy pht sinh chng loi AMF

(http://www.ffp.csiro.au/research/mycorrhiza [62])

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1.3. PHN LOI BO T AMF

Thng thng, vic phn loi nm ni cng sinh ch yu da vo cc

c im hnh thi v cu trc (Bng 1.1). Mt trong nhng c s quan trng

phn loi theo hnh thi l bng mu ca Morton. Phng php ADN v

gii phu s c dng nh gi quan h mc cao hn do Smith v

Tinker xut 1997 [51].

Nhn chung, h thng phn loi AMF ang p dng da trn c s h

thng phn loi ca Morton v Benny a ra vo nm 1990 v c hon

chnh dn nh hng lot cc nh nghin cu tip [39].

Lch s hnh thnh h thng phn loi ny trc ht phi k n vic

hnh thnh chi Endogone nm 1808. Tip l chi Glomus do anh em

Tulasne m t nm 1844. n nm 1849, tc gi Fries xy dng nn h

Endogonaceae, sau h ny b thay i do loi mi pht hin c rt t c

im chung. y l thi im hon thin s phn loi v phng php

nhn bit tt c cc loi bo t ca AMF.

Nm 1959, Moss (nh Gii phu thc vt) v Bowen (nh Sinh thi hc)

a ra h thng m t da trn cu trc vch t bo, mu sc v c im

t bo cht [40]. Tuy nhin, khi p dng phng php ny th Gerdermann

pht hin ra rng Endogone c s lng loi rt ln, cn phi xem xt li v

tc gi chia Endogone thnh 7 chi vi 3 chi khng cng sinh: Endogone,

Modicella, Glaziella v 4 chi cng sinh: Glomus, Sclerocystics, Gigaspora,

Acaulospora (trong Gigaspora v Acaulospora l 2 chi mi) [31].

Vi h thng phn loi ca Gerdermann, Vin Nghin cu sinh hc

Vin Hn lm khoa hc n phn lp t t vn m 4 chi AMF:

Glomus, Sclerocystics, Gigaspora, Acaulospora v 1 chi khng cng sinh

(Endogone).

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Nm 1982, Trappe v Schenck xut a 5 loi AMF ra khi chi

Sclerocystics hnh thnh chi mi Scutellospora [58], n nm 1987 Walker

cng xut nh trn [60]. Nm 1990, tc gi Morton v Benny t 5 chi ca

Walker vo 3 h: Glomaceae, Gigasporaneae, Acaulosporaceae v 2 b ph:

Glomineae, Gigasporineae, trong 2 b ph ny c t trong b mi

Glomales [39].

Nm 1998,Trung tm Nghin cu AMF ca i Loan (Arbuscular

mycorrhizal fungal Collection center in Taiwan ACT) ngh cng nhn 2

chi mi l Glomites v Jimtrappea. Hin nay, h thng phn loi ca ACT

thng c s dng cc nc chu .

Nm 2008, Shipra Singh v tg tip tc cng b thm 2 h AMF l:

Archacosporaceae v Paraglomaceae vi 2 chi mi l: Archacospora v

Paraglomus [49].

1.4. CC PHNG PHP NGHIN CU MYCORRHIZA

Phng php tch bo t

Mun tin hnh phn loi th cng vic u tin l tch bo t t t.

Thng thng, bo t c tch bng sng t v lc. Phng php ny c

s dng lc tuyn trng t t do Gerdermann ci bin cho ph hp vi

nm ni cng sinh [32]. Trn c s nhm tc gi Daniel v Skipper (1982)

v tip sau tc gi Tommerup (1992) ci tin thnh phng php sng

t (wet sieving) qua ry kt hp vi ly tm trong thang nng ca sucrose

(dch 50%) [25, 56].

Quan st, m s lng bo t

Ty vo kch c, qu trnh quan st bo t c th c thc hin trn

knh lp hoc knh hin vi c phng i nh. S lng bo t c xc

nh bng phng php m trc tip trn mng lc c chia ca hng

Satorrius [24].

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Xc nh dng xm nhim

xc nh cc dng xm nhim, ngi ta tin hnh nhum Tryphan

blue (0,005%) lm bin mu th cn xc nh trong r ca cy ch, do Philip

v Hayman xut c th nh sau: u tin, nm r cng sinh c ra

nhiu ln bng nc ct nhm loi b t v cc tp cht hu c bm trn b

mt ca r sau un nng bng dung dch KOH 10% nhit 90oC trong

vng 2h, sau ra bng axt v bin mu bi Tryphan blue, kt qu l si

nm s bt mu xanh [43]. i vi thc vt c nhiu sc t trong r,

Kormanik v cng s xut phng php s dng fuschin lm mt mu

nhng mu r cy cn quan st [35]. Nm 1996 tc gi Brundrett a ra

mt phng php mi vi thuc nhum l Chlorazol black E, nh c th

quan st mt cch r rng nhng giai on ca AMF trong r cy ch [24].

Tuy nhin, tt c cc phng php trn u c chung mt nhc im l

tn thi gian v gy ph hy mu. Mt khc, qu trnh v mc bin i

mu l khc nhau vi tng mu r. a s cc loi thuc chi Gigaspora v

Scutellospora bin i mu rt mnh vi Tryphan blue m khng ph thuc

vo loi cy ch theo Morton (1988) [38]. Nhng c loi ch bin mu trung

gian vi Tryphan blue nh Acaulospora trapei [18]. Thm ch, mt s loi

ca chi Glomus (G.leptoticum, G.maculosum) hoc loi Acaulospora

myriocarpa li khng bin mu vi Tryphan blue [38].

Mc cht ch ca mi quan h gia thc vt v AMF c th hin

thng qua s lng bo t c trong t. Tuy nhin, vic xc nh s lng bo

t i khi gp nhiu kh khn. gii quyt vn , ngi ta thng dng

mt ch s trung gian l h s xm nhim. Phng php n gin l ct h

thng r thnh nhng mu nh v xc nh t l Mycorrhiza. Tuy nhin,

phng php ny khng hiu qu khi t l Mycorrhiza ln. Nm 1975, da

trn c s phng php ng cho ca nhm tc gi Newman, Sparling v

Tinker ln u tin p dng vi AMF, sau c dng so snh vi cc

phng php xc nh khc. Hin nay, phng php ny rt thng dng [52].

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1.5. S PHT TN V NH HNG CA AMF I VI THC

VT CH

Nm 1991, tc gi Friese v Koske cho rng tt c bo t ca AMF u

phn tn mt cch th ng vi nhng nhn t tch cc l gi v ng vt

[29]. Nhng theo MacMahon v Warner th nhng vng kh hn, gi l nhn

t quan trng nht to ra s ly nhim t nhin ca AMF [36]. Ngc li, vi

nhng ni m t, ng vt li l nhn t ng vai tr ch yu [29].

Nm 1983, Hayman cho rng s lng bo t AMF trong t l ch tiu

quan trng nh gi mc u th ca loi. Trong t canh tc, s lng

loi v s lng bo t nhiu hn trong t t nhin [34]. Mt khc theo tc

gi Friese th AMF khng d pht tn nn tng t canh tc l v tr tt nht

xc nh s lng bo t [29].

ng thi vi nhng nghin cu v phn loi, tch bo t, cu trc

AMF th nh hng ca AMF i vi s sinh trng ca cy cng c quan

tm t rt sm.

Nm 1963, tc gi Gerdemann s dng t khng kh trng nh

mt cch ly nhim AMF v chng minh c rng cy trng s pht trin

nhanh khi c mt AMF [32]. Nm 1959, trong mt bo co khoa hc khc

ch ra rng, vic nhim AMF lm tng sinh trng ca cy to con t chi

[40]. Cho n nm 1968, Gerdemann tin hnh th nghim trn cy ng v

yn mch cng cho kt qu tng t [30].

Nh vy, trong nhiu nm qua, nhng nghin cu v AMF ch tp

trung vo vn nh hng ca chng i vi sinh trng cy trng m

khng bit rng gi tr AMF mang li cho thc vt ch cn ln hn nhiu.

Nm 1968, bo t nm ni cng sinh c th sng cng vi thc vt trong

chu v a ra phng php nhn nui th nhng nghin cu v AMF v nh

hng ca chng i vi thc vt mi c tin hnh su rng trn nhiu lnh

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vc nng lm nghip [30, 41]. Cc nh khoa hc chng minh rng: AMF

khng ch lm tng kh nng sinh trng, pht trin ca cy trng m cn c

th lm tng kh nng hp thu khong (nh phtpho, ng, km) trong t;

lm gim mc sc ca cy khi t b nhim mn, t qu m, nhit

t cao v nhiu nguyn nhn khc.

V kh nng bo v cy ch chng li cc tc nhn gy bnh ca AMF,

Schonbeck v Dehne (1989) nghin cu trn 11 loi cy trng ph bin l

u, la mch, la m, c rt, ng, hnh, thuc l, c chua, da chut, rau

dip, h tiu nhn thy, chng lm gim 40% cc bnh r thng gp

trn cc loi cy ch ny [48]. Nm 2000, tc gi Ted cng ch ra kt qu

tng t khi nghin cu kh nng chng bnh cc cy nhit i c AMF

(gim 30 45% cc tc nhn gy bnh) [54].

Nm 2002, bng thc nghim, Bali v tg, chng minh hiu qu ca

AMF i vi bnh ho cy bng do nm Fusarium gy ra. i vi bnh r

gy ra bi tuyn trng, AMF c th ci thin sc sng ca cy ch, t hn

ch nhng thit hi v sn lng, c bit vi nhng vng t c hm lng

P2O5 thp v cy c nhim AMF trc khi b nhim tuyn trng. Nh vy,

AMF c th lm gim ngun bnh hoc gim nh hng ca bnh r c

nguyn nhn do nm v tuyn trng gy ra. Tuy nhin, tc dng ca AMF

khng th hin r i vi bnh l v bnh do virus [20].

Khi nghin cu s lng AMF trong t, Schuybert v tg nhn thy

rng: cc loi t khc nhau, s lng bo t AMF l khc nhau [50].

Nm 1989 tc gi Schonbeck phn lp c 15 loi thuc 3 chi khc

nhau l: Glomus, Sclerocystis, Acaulospora trong t vng r ca cy nho v 7

loi thuc chi Glomus trong t vng r ca cy to [48]. Trong t trng trt

thng xuyn (t trng la nc, u, la m v nhiu loi cy trng khc)

lun c s lng bo t AMF cao hn nhng thnh phn loi ca AMF li thp

hn so vi trong t t nhin. Nm 1989 Schonbeck v Dehne nghin cu v

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mi quan h gia AMF vi cc vi sinh vt vng r, cho thy s c mt AMF

lm tng ng k lng vi khun tng s (c bit l nhm Pseudomonas) [48].

Khi nhim 3 loi Gigaspora margarita, Glomus macrocarpum v

Glomus caledonium cho cy du ty, lm tng ng k sinh khi v hm

lng phtpho trong cy, trong , Gigaspora margarita c hiu qu kch

thch mnh hn 2 loi cn li. Nm 1982, Dehne cng pht hin tc dng kch

thch sinh trng ca AMF trn cy hnh v cy ng [26]. n nm 1989,

nghin cu khc cn pht hin thm bn cnh kh nng tng sinh khi, tng t

l thn/r th vic nhim AMF cn lm tng hot ng ca enzyme

nitrogenase v tng mc ng ho phtpho ca cc cy h u [59]. Ngoi

ra, Sung v tg cn chng minh c rng: Thng qua hot ng trao i cht

ca mnh, AMF c nh hng n Pyrophotphat (PPi) trong cy ch, iu ny

cho thy tc dng rt ln ca AMF i vi ton b qu trnh sinh trng v

pht trin ca cy ch [48].

Nm 2004, Trn Vn Mo nghin cu hiu qu ca nm VA

Mycorrhiza ch yu l nm Glomus, v kh nng hp thu dinh dng P. Hm

lng P trong t bo r cy bp c s cng sinh ca nm VA-Mycorrhiza tng

35% i vi cc loi nm Glomus mosseae v 98% i vi loi nm Glomus

fasciculatum. Hm lng P c tch lu trong r bp dng hn hp phn t

P hu c v acid ho tan. Hng ngy P c di chuyn n cc b phn ca

cy bp theo phng php ng lc hc, v ph thuc vo tng giai on sinh

trng ca cy [7].

Nh vy, cc kt qu nghin cu nh hng ca AMF i vi s sinh

trng v pht trin ca cc loi cy trng khc nhau cho thy chng u c

tc dng tng sinh khi, tng qu trnh trao i cht v thu nhn cht dinh

dng, tng hot ng ca cc enzyme.

Tm li vai tr cng sinh ca Mycorrhiza i vi s sinh trng, pht

trin ca cy trng c khi qut nh sau [52]:

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* Kh nng huy ng nc v cc cht dinh dng: H si nm cng

sinh xung quanh vng r lm tng din tch b mt tip xc, tng kh nng ht

nc v cc cht dinh dng. i vi cc cht dinh dng km di ng (nh

ion phtphat, ng, km), cy trng ht cc ion ny nhanh hn kh nng

khuch tn ca chng trong dung dch t nn thng hnh thnh vng hp

cn kit xung quanh r. Khi , h si nm nhanh chng di ra, vt qua

vng cn kit n vi ni c phtpho d tiu. Do c ng knh nh

hn so vi lng ht ca r, si nm c th len li khp ni trong t, k c cc

l hng rt nh m r khng qua c ht dinh dng cung cp cho cy.

i vi phtpho b c nh cht, si nm cng sinh s tip cn v tit ra cc

axt hu c qua phn ng ca anion hu c phn t nh (nh oxalate)

sau c th i ch cho phtpho (b ht cht b mt bi hydroxide kim loi)

bng phn ng trao i, ho tan oxide kim loi, to phc kim loi trong dung

dch, do vy ngn chn c s kt ta ca phtpht kim loi. Nm cng sinh

Mycorrhiza cn gii phng phtpho v c thng qua khong ho cht hu c

(thu phn hp cht ester phtpht hu c). Cc hnh thc cng sinh Ericoid

Mycorrizha v Ectomycorrizha cn c vai tr quan trng trong vic khong

ho nit. Ch cn mt lng nh AMF c th huy ng dinh dng t khi

lng ln xc thc vt c t l C/N cao (hm lng lignin v tannin cao).

* Dng chy cacbon trong cy cng sinh Mycorrhiza: Dng cacbon c

th ch theo mt chiu cng bc t cy xung AMF hoc cng c th theo

chiu tho thun do AMF t phn gii hp cht giu cacbon t. Dng chy

cacbon t cy xung t khng lm cy trng thiu ht cacbon v khi AMF

xm nhim vo r cy s kch thch qu trnh quang hp mnh hn gp bi

(tr trng hp nh sng qu yu). Trong h sinh thi, dng chy cacbon

xung nm v t c mt s vai tr quan trng sau:

- Si AMF sn sinh ra cc enzyme thy phn (nh protease,

phosphatase) c vai tr quan trng trong qu trnh khong ho cht hu c

v huy ng cht dinh dng cho cy trng.

15

- Si nm ko di lm tng lin kt vi cc ht t, ci thin cu tng

t, thng thng c n 1 20 m si nm/gam t.

- To nn cng ng vi sinh vt vng r. y l ch tiu quan trng

nh gi ph nhiu ca t.

* Tng kh nng chng chu hn: trong mi trng t kh nm r gip

cy hp thu nc bng cch tng cng tc thot hi nc so vi nhng

cy khng c nm r cng sinh. Allerm cho rng tc dng ca nm trong vng

kh hn biu hin ch yu l lm tng tnh chu hn ca cy v tng nhanh tc

truyn nc trong cy. Nm 1982, Berea v tg li cho rng tc dng ca

nm r l ci thin kt cu t v nng cao lng nc trong t t lm

tng kh nng hp thu nc cho cy [21].

* Gip cy chng chu vi bnh hi: nm cng sinh r cy c tc

dng bo v cy chng li mt s vi sinh vt gy bnh nh Phytophthora

infestans (mt loi to tng t nm). Do Phytophthora infestans khng th

xm nhp qua h si nm lt vo r [10].

- Ngn chn c hc s xm nhp ca ngun bnh bng cu trc si nm

an xen trong r cy.

- Sn sinh cc hp cht khng sinh (antibiotic).

- Cnh tranh dinh dng vi vi sinh vt gy bnh, gp phn lm tng sc

khng cho cy ch.

1.6. TNH HNH NGHIN CU AMF TRN TH GII BNG K

THUT SINH HC PHN T

Trn th gii c nhiu nghin cu v nm Mycohriza mc phn

t nh RFLP, RAPD khuych i trn nhiu vng khc nhau ca nm c

thng k theo bng 1.2. Tuy nhin vic phn loi bo t bng sinh hc phn

t vn cn nhiu hn ch do kh nng thu nhn DNA tng s t bo cn thp.

16

Bng 1.2. Kt qu nghin cu AMF bng sinh hc phn t [45]

Vng

gen

K thut sinh hc phn t

on mi T bo ch Ti liu

tham kho

SSU

rDNA

Genomic

DNA

PCR PCRRADP

VANS1

OPA-02 v OPA-04

OPA-18 v P124

OPA-18 v P124

Glomales

Glomusversiforme

Gl. mosseae . Gl. Caledoniu

Acaulosporalaevis

Gigasporamargarita

Scutellospora gregaria

109

39

SSU

rDNA PCR VANS1 v NS21 G. intraradices 83

Genomic

DNA

Competitive

PCR PO v M3 G. Mosseae 46

ITS PCRRFLP ITS1 v ITS4 Glomus sp., Scutellospora

sp. 41

SSU

1492 PCR NS71 v SSU1492 Gigaspora sp. 75

Partial

rDNA PCRpartial

SS38 v VANS1

VANS1

VAGIGA

R v bo t ca AM,

Scutellospora v Glomus

Gigasporaceae

30

ITS1 and

ITS2 PCR ITS1 v ITS2 G. margarita 32

ITS PCR ITS1 v ITS4 G.mosseae v Gigaspora

margarita 29

SSU

rDNA

PCR-RFLP

PCRnested

LR1 v FLR2

FLR2-5.23 v

FLR2-8.23

LR1-23.46

Di nhm ca Glomales

G. mosseae, G. intraradices

G. roseae

45

28S

rDNA PCRSSCPs LSU-Primers Glomus sp. 82

SSU

rDNA PCR NS31 v AM1 Glomus sp. 4

17

SSU

rDNA

PCRSSCP

Nested-PCR

VANS1

ITS, AM1

Di nhm ca Glomales

60

ITS PCRRFLP ITS1 v ITS4 G. mosseae 74

ITS Nested PCR

SSCP

Eukaryotic

universal primer

Glomus sp.

Glomus-specific ITS primer 110

ITS Nested-PCR ITS 5 v ITS4 Glomeromycota (except

Archaeosporaceae) 44

ITS PCR

SSU-Glom/LSU-

Glom 1

ITS5 v ITS4

Cc nhm chnh vi Glomeromycota

44

1.7. TNH HNH NGHIN CU VIT NAM

Vit Nam, Mycorrhiza c nghin cu t nhng nm 60 ca th k

XX nhng n nay mi t c mt s kt qu nghin cu v nm ngoi

cng sinh, hu nh cha c thnh tu trn nm ni cng sinh, c bit l vi

cy cam.

Nghin cu u tin v nm cng sinh trong lm nghip l vic s dng

lp t tng mt ca rng thng gieo m cy con - y c th c coi

nh mt hnh thc nhim nm t nhin ca Lm Cng nh v c s

dng nh mt bip php lm sinh trong mt thi gian kh di [2].

Sau l cc kt qu nghin cu ca Nguyn S Giao (1976) v s c

mt ca nm ngoi cng sinh r cy thng. Nghin cu s dng nm cng

sinh thun chng to r nm cho cy thng con, kt qu cho thy s vt

tri v chiu cao v ng knh ca nhng cy c nhim nm so vi cng

thc i chng l 20 30% [1]. Bn cnh , Nguyn S Giao v Nguyn

Th Nhm (1980) cn nghin cu s dng nm ni cng sinh phng bnh

vng ci thng con v cng t c nhng kt qu nht nh [3].

T nm 1970 1980, Vin Nghin cu lm nghip (nay l Vin Khoa

hc Lm nghip Vit Nam) tin hnh phn lp v nui cy thun chng

18

nm cng sinh [3]. Mt vi th nghim c tin hnh trn t cn vi cy

ch l mt s loi thng nhp ni. Tuy nhin, nhng th nghim ny sau

phi dng li v nhiu l do. Nhng nghin cu ng dng nm cng sinh

trong lnh vc lm nghip trong giai on tip theo b ngng tr [15,16].

Nm 1998, Phm Quang Thu v tg tip tc nghin cu v nm cng

sinh vi thc vt, kt qu l xc nh c 37 loi nm (thuc 9 h v 7 b)

cng sinh vi 3 loi thc vt (thng nha Pinus merkussi, thng ui nga

Pinus massoniana v thng caribe Pinus caribaea). Cng trnh ny c coi

nh s khi u li cho nhng nghin cu v nm cng sinh vi thc vt, c

bit trn i tng cy lm nghip. Kt qu khng ch dng li vic xc

nh thnh phn loi nm m cn i su hn trong vic nghin cu sn xut

cc ch phm nm cng sinh, ng dng rng ri trong gieo m cy con

keo v bch n. y l mt mc quan trng trong nghin cu ng dng nm

cng sinh v ch ng c ngun nm ly nhim thng qua cc ch phm

sinh hc. Gi tr ca nghin cu ny cn c th hin trong thc tin sn

xut ti tnh Vnh Phc, s dng ch phm nm cng sinh ci thin ng

k tnh hnh sinh trng ca cy trng nhiu vng t i [14]. i vi

AMF vi cy dc liu, nghin cu u tin Vit Nam c cng ch dng

li thu thp mu bo t nm cng sinh [5].

Mt iu ng mng cho khoa hc Vit Nam v nm cng sinh ni

chung v AMF ni ring ti Hi tho v nm cng sinh u tin ca Vit Nam

c t chc ti Vin Th nhng nng ho vo nm 2004 vi s gp mt

ca cc nh nghin cu c bn thuc i hc Quc gia H Ni v cc ngnh

nng nghip, lm nghip, dc hc... Tuy cc tham lun ti Hi tho ny cn

mang tnh l lun, t cp n cc kt qu nghin cu nhng nm ni cng

sinh c ch nh mt ni dung chnh ca Hi tho, mt s nh khoa

hc cn xut nh hng nghin cu AMF trong nhng nm tip theo.

19

Nm 2005, nhm nghin cu ca Nguyn Vn Sc v tg [12], khi tin

hnh nghin cu Nm r ni cng sinh (Arbuscular Mycorrhiza Fungi) v

qun th vi sinh vt trong t trng bi c sn oan Hng, Ph Th

pht hin s c mt ca AMF trong tt c cc mu thu thp c nhng t l

xm nhim thp ch t mc 3/5 [33], kh nng ny mm ca cc bo t nm

r bi thp (ch t 16%). Trong mt nghin cu khc, cc tc gi s

dng 3 loi cy k ch (ng, cao lng, la mch) v 3 chng AMF nhn

bo t ri a ra kt lun: Khi nhn bo t nh cy k ch, mi loi cy k

ch thch hp cho mt chng nm, ring cao lng khng thch hp dng lm

cy k ch nhn nhanh bo t AMF, thi gian thu bo t tt nht l 25

40 ngy sau khi cy k ch mc [13].

Cc kt qu nghin cu gn y ca Nguyn Th Minh (2005, 2007)

trn cy h u cng cho mt s kt qu kh quan ban u [8, 9]. Cng nm

2007, Nguyn Hong Yn cng b cng trnh nghin cu u tin v phn b

ca AMF trong cy h Sao du ng Nai [17].

Tuy c nhiu ti liu cp n vai tr ca nm cng sinh AMF nhng

cha c nghin cu v vai tr ca nm cng sinh i vi cy cam cng nh

nh hng ca cc bin php k thut canh tc, dinh dng t n nm cng

sinh trn cy cam.Cc nghin cu su rng v AMF mc sinh hc phn

t, bng cc phng php nh RFLP, RAPD cha c tin hnh hoc cha

c cng b.

Tm li, mi quan h cng sinh nm r thc vt t c quan tm

nghin cu v ng dng Vit Nam, c bit cha c cng trnh khoa hc

no nghin cu v AMF cy cam. Bi vy ti: Nghin cu h nm cng

sinh (Arbuscular Mycorrhiza) trong t trng cam Qu Hp Ngh An

c thc hin nhm gp phn thit thc vo hng nghin cu mi: ng

dng AMF nng cao nng sut cy cam ni ring v cy trng nng lm

nghip ni chung trn c s bo v mi trng sinh thi theo hng pht trin

bn vng trong th k XXI.

20

CHNG II

NGUYN LIU V PHNG PHP NGHIN CU

2.1. NGUYN LIU

2.1.1. Mu

S dng 60 mu t v r c ly cc tng t khc nhau (0 20 cm, 20

40 cm, 40 60 cm) ti t trng cam X Minh Tn - Qu Hp Ngh An.

Thi gian ly mu vo thn 8 v thng 10 nm 2011.

Bng 2.1: Trnh t mi dng cho phn ng PCR

MI Trnh t nucleotide

VANS1_5 5' GTC TAG TAT AAT CGTTAT ACA GG 3'

NS21 5AAT ATA CGC TAT TGG AGC TGG 3

2.1.2. Ha cht v thit b nghin cu

Ha cht:

Cc ha cht dng cho phn ng PCR (buffer, dNTP, enzyme taq

polymerase) do hng Fermentas PureXtreme (M) cung cp v b Kit tch

chit DNA, tinh sch DNA nhp t hng QIAGEN (Canada). Ngoi ra, mt

s ha cht khc cn c chng ti s dng nh: EtBr, gel agarose, dung

dch m TAE 1X

Thit b ch yu:

- B ry vi cc kch c: 10.000 m; 70.000 m; 5.000 m; 2.500 m;

1.000 m; 750 m; 500 m; 250 m; 90 m v 40 m.

- Knh hin vi quang hc Olympus BH vi h thng chp nh chng

rung.

- B vi gp v b ht chn khng p sut thp.

- Phu ht km giy lc Whatman s 1 4.

21

- T lnh -200C Vestfrost (an Mch), My li tm BioFuge fresco

(c), L vi sng Sharp (Nht Bn), Cn in t Precisa (Thy S),

My chu trnh nhit GenAmp PCR System 9700 (M), My chp gel

BioRad (c), T lnh Toshiba (Nht Bn), My in di Mupid

2plus (Nht Bn), My li tm nhanh Picofuge (M), My gii trnh t

ABI 3100 Bio System (M), Pipette Gilson (Php), B n nhit

Memmert (Hn Quc), B dng c in di (i Loan), My o quang

ph (c).

2.2. PHNG PHP NGHIN CU

2.2.1. Phng php tch bo t

- Ly mu, bo qun mu: Theo phng php ca Hayman 1983, mu

ly ba tng phu din khc nhau: Tng 0 - 20 cm, tng 20 40 cm v tng

40 60 cm. Mu sau khi ly c bo qun 4oC cho n khi phn tch [34].

- Tch bo t t t: S dng k thut sng t (wet siewing) qua ry

kt hp vi ly tm trong thang nng ca sucrose (dch 50%) theo Daniel&

Skipper (1982) [26], Tommerup (1992) [56].

Bc 1:

+ Cn mu t 50 g

+ Loi b cc mnh rc th v trong mu t

+ Sau cho lng t cn vo trong nc t nht 30 pht trc khi

sng, cc khi t to phi c p v.

Bc 2:

+ t c trn u trong ca dung tch 1lt nc, sau trong 10 pht

cho cc mu t ln lng xung ri gn dch sang mt lot sng c kch thc

khc nhau.

22

+ Qu trnh ra v gn lc c lp li nhiu ln cho ti khi nc trong

r v cc mu rc th c gi li trn sng c kch thc ln nht, cn bo

t c gi li trn sng c kch thc b nht.

Bc 3:

+ Thu li cc vt cht bm trn sng c kch thc b nht sau chuyn qua

cc ng ly tm, tin hnh ly tm ln 1 5000 vng/ pht trong thi gian 5 pht.

+ Sau bc ny cc cn l lng v rc c loi b.

Bc 4:

+ Cho 50 l dung dch sucrose 50% vo ng ly tm ri lc mnh cc ng

(Hnh 2.5)

+ Tip tc ly tm tc 11000 vng/ pht trong 2 pht tch bo t

t thnh phn t. Sau khi ly tm, bo t nm trong dung dch huyn ph

sucrose.

Bc 5:

+ Ht dch huyn ph sucrose c cho ln giy lc v t trn phu

Buchner trc khi lc ht chn khng (dng giy lc Whatman GF/A s 1-4).

+ t giy lc cha bo t vo a petri ri em quan st bo t di knh

hin vi in t.

Bc 6:

Bo t sau khi c pht hin di knh hin vi c chp nh. S

lng bo t c xc nh bng phng php m trc tip trn mng lc c

chia ca hng Satorri.

- Xc nh hnh dng v kch thc ca bo t: Bng so snh ca

Morton [38].

- Xc nh cc dng xm nhim AMF: Nhum Tryphan blue c ci tin

ca Brundrett Mark v cng s [25].

- Xc nh tn chi v loi: Theo Schenck v cng s [47].

23

- Gi ging cc bo t AMF: Ly nhim ch ng vo chu cha t ct

kh trng trc khi trng cy k ch ng [18].

- Mu sc ca bo t: Xc nh bng bng mu chun 4 nhn t.

24

Hnh 2.1. Bng mu so snh phn loi nm Mycorrhiza

25

2.2.2. Tch chit DNA tng s

Qu trnh tch chit DNA tng s t mu r c ci tin theo phng

php tch DNA tng s ca White v cng s (1990) [61].

Phng php ny bao gm nhng bc chnh sau:

Bc 1: R c ct nh cho vo ng effendorf 1,5 ml.

Bc 2: Dng pipette ht 300 l dung dch m, cho vo ng

effendorf c cha mu.

Bc 3: Dung dch c chy qua 3 chu k nng lnh:

Lm nng 65 C/ 15 pht

Lm ng lnh b / 15 pht

(Hai chu k ny xen k nhau v c lp li 3 ln)

Bc 4: Sau dng chy gi nh v em i nhit 65 oC/30

pht.

Bc 5: B sung 400 l Clorofom vo dung dch sau vortex.

Bc 6: Dung dch c ly tm 10000 vng/pht trong 30 pht.

Bc 7: Sau khi ly tm dch chia lm 2 pha, thu pha trn chuyn

sang ng effendorf mi v ta bng dung dch Isopropanol lnh.

Bc 8: Ly tm lnh 12000 vng/pht trong 30 pht.

Bc 9: Loi b dich, thu cn v ra cn bng ethanol 70% lnh.

Bc 10: Sau khi ra sch, lm kh v b sung 20 l m TE, ri

bo qun t lnh -20 oC.

Sau , sn phm DNA tng s c tin hnh in di kim tra sn

phm trn gel agarose 0,8%.

2.2.3. in di DNA trn gel agarose

in di l k thut tch cc phn t da trn in tch, kch thc v hnh

dng ca chng trn gel. Trong phng php in di trn gel, cc phn t

26

DNA c in tch m, c ko qua mt lp gel bn lng nh mt dng in

v pha cc dng trong bung in di.

Thng thng, gel c cha mt phn ng ca thch tinh khit gi l

agarose, cht ny ngoi vic lm gel ng nht hn so vi thch m n cn

hot ng nh mt ry phn t, lm tch bit cc on da trn kch thc

ca chng khi trong in trng.

Cc on DNA cng nh s chuyn ng cng nhanh v cng xa so

vi cc on ln. xc nh kch thc ca mt on DNA, ngi ta so

snh khong cch n so vi khong cch m cc on DNA tiu chun (l

mt tp hp nhiu on DNA bit r kch thc - marker) i qua.

Nng agarose c s dng khc nhau ty thuc vo th nghim

cn nghin cu, thng nm trong vng t khong 0,8 - 1,5%.

hin hnh cc bng DNA trn bn gel, ngi ta thng s dng

thuc nhum ethydium bromide (EtBr). Cht ny lin kt vi DNA bng cch

xen vo gia v k st vi cc cp baz (lin kt nhum). Khi chiu nh sng

(tia UV) vo bn gel, cc lin kt nhum s hp th v pht hunh quang, kt

qu c th nhn bit c bng mt thng v chp nh c.

Quy trnh:

* Chun b gel agarose: cn 0,8 g agarose cho vo 100 ml dung dch m

TAE 1X. un trong l vi sng cho agarose tan hon ton. ngui xung

khong 50 oC ri dung dch agarose vo khay in di c ci sn lc. Sau

khong 30 pht, g lc ra v t bn gel vo b in di. m TAE 1X

vo b dung dch ngp cch mt gel 1 2 mm.

* Tra mu DNA: Ly mu khong 5 10 l mu DNA trn vi 3 l m

loading dye (cht ny va c tc dng to mu quan st, va c tc dng

gip cho mu DNA lng xung y ging trc khi chy in di) v tra vo

cc ging nh trong gel. S dng marker 1kb lm ch th phn t.

27

* Chy in di hiu in th 100V trong 30 pht. DNA di chuyn t

cc m n cc dng. Quan st s di chuyn ca mu bromophenol blue

bit khi no dng in di.

* Nhum bng EtBr: bn gel c ly nh ra khi khun v ngm vo

dung dch EtBr nng 2 l/ml trong thi gian khong 10 pht. Sau ly

bn gel ra trng qua nc ri quan st v chp nh di nh sng tia t ngoi

ca my soi chp gel (Bio Rad).

2.2.4. K thut PCR

PCR l ch vit tt ca Polymerase Chain Reaction, c dch l phn

ng chui trng hp hay phn ng khuch i gen. My chu trnh nhit (my

PCR), cc chui DNA mong mun c nhn ln hng triu ln trong thi

gian ngn nh hai on mi oligonucleotide tng t hai u 3 hai ca

on DNA ch vi s tham gia ca Taq DNA polymerase.

Qu trnh PCR l mt chui nhiu chu k ni tip nhau, mi chu k

gm 3 bc (bin tnh, gn mi, ko di) lp i lp li nhiu ln, mi ln s

lm tng gp i lng mu ca ln trc. PCR l mt k thut ph bin

trong sinh hc phn t nhm khuch i (to ra nhiu bn sao) DNA m

khng cn s dng cc sinh vt sng nh E. coli hay nm men. PCR cng l

mt phng php thng dng hin nay pht hin cc bnh di truyn.

Cc phn t DNA nhng t bo hot ng bnh thng ch c

nhn ln trong qu trnh phn bo nguyn nhim. thc hin c qu trnh

ny, i hi phi c mt mt enzyme DNA polymerase, cng nh s tham gia

ca cc on mi c trnh t b sung gn vi si DNA khun c u 3OH t

do v cc dNTP lm ngun cung cp cc nucleotide. Trong qu trnh phn

bo nguyn nhim, cc si DNA kp c m xon v tch thnh DNA si

n. Di tc dng ca DNA polymerase, qu trnh tng hp DNA c xy

ra theo cch gn ln lt cc nucleotide vo on mi ti v tr 3OH ko

di chui theo nguyn tc b sung vi DNA khun.

28

Nguyn tc ca PCR l s dng DNA polymerase chu nhit tng

hp trong ng nghim cc DNA mi, t mch khun trong mi trng d

tha dNTP v cc cp mi c hiu. Cc on DNA mi hnh thnh li c

s dng lm khun tng hp cho cc chu k tip theo. Nh vy s lng

bn sao s tng gp 2 sau mi chu k v sau n chu k, tnh theo l thuyt s

lng bn sao l 2n.

K thut PCR yu cu cc thnh phn sau:

+ DNA khun mu.

+ Mi c hiu chiu di 15 - 30 nucleotide.

+ Bn loi deoxyribonucleotide triphosphate (vit chung l dNTP).

+ Taq DNA polymerase chu nhit.

Nhn on gen SSU bng PCR

Bng 2.2 Thnh phn phn ng PCR

- Thnh phn phn ng Nng phn ng Th tch (l)

Nc kh ion v trng 11,75

Buffer 1 X 2,5

dNTP 10 mM 0,5

Mi xui 10 pmol 1

Mi ngc 10 pmol 1

Taq DNA polymerase 1 U 0,25

DNA khun 8

Tng 25

29

Chu trnh nhit c ti u ha: Cho phn ng PCR

Hnh 2.2. Chu trnh nhit PCR

Chu trnh nhit trong k thut PCR c thc hin trn my GenAmp

PCR System 9700 thng qua 3 giai on (Hnh 2.9):

Giai on 1: Giai on tin bin tnh, tho xon DNA 950C trong 3

pht.

Giai on 2: Giai on khuch i on DNA thng qua 3 bc. Mt l

bc bin tnh DNA 950C trong 1 pht, hai l bc gn mi 500C trong 1

pht v ba l bc ko di chui 720C trong 2 pht 30 giy. Qu trnh ny

lp li trong 30 chu k.

Giai on 3: Giai on tng hp nn chui DNA mi 720C trong 10

pht, sau mu c bo qun 40C.

2.2.5. Tinh sach san phm PCR

Sau khi tin hnh in di cc mu PCR lng ln, phn gel cha on

gen mong mun c thu li vo ng effendorf 1,5 ml. Qu trnh tinh sch

bao gm nhng bc sau:

Bc 1: Cn ng effendorf cha gel thu.

30

Bc 2: B sung Binding buffer II vo ng, ch c 100ng gel cn b

sung thm 400 l buffer (V d: 125ng gel th cn thm 500 l buffer), sau

em ng trong b n nhit 65oC n khi gel tan hon ton.

Bc 3: Cho ton b dch trong ng effendorf vo ct li tm lng trong

ng EZ 10 (loi ng chuyn dng cho li tm) v im trong 2 pht nhit

phng. Sau 2 pht, em ng i li tm 12000 vng/pht trong 3 pht. Kt thc

li tm, loi b ht phn dch y ng, thu ct.

Bc 4: Chuyn ct sau li tm trc vo ng EZ 10 mi, b sung

Wash Solution vi lng ti a 800 l v em i li tm lnh 12000 vng/pht

trong 2 pht. Cng ging nh bc 3, b phn dch y ng, thu ct.

Bc 5: B sung 500 l Binding buffer nu nh mu mun tin hnh c

trnh t lun.

Bc 6: B sung 700 l wash buffer tip tc li tm lnh 12000 vng/ 1

pht.

Bc 7: ly tm li mt ln na loi b ht dch.

Bc 8: chuyn ct t bc 7 sang ng 1,5 ml kh trng. B sung 50

l Elution Buffer tip tc li tm lnh 12000 vng/ 1 pht.

Bc 9: DNA sau khi c tinh sch cn phi c bo qun -20 oC,

dng 3 - 5 l mu i in di kim tra, chy in di bng gel agarose 0,8%

trong 30 pht, sau nhum bng EtBr trc khi chp nh di tia UV trn

my Bio Ra.

31

CHNG III

KT QU V THO LUN

3.1. PHN LOI AMF TRONG CC MU NGHIN CU

3.1.1. Hnh thi v cu to bo t AMF trong t trng cam Qu Hp - Ngh An

Trong tng s 60 mu c kho st ti t trng cam Qu Hp Ngh An nm 2011 c tng s 16 kiu hnh

bo t AMF thuc 6 chi c tm thy nh bng 3.1.

Bng 3.1 Kt qu phn loi bo t AMF

Tn chi Tn chng Kch

thc m

c im Hnh dng

Acaulospora

Appendicula 150-175

Bo t hnh cu hoc gn hnh cu. a

s bo t mu trng, mt s t bo t

mu nu hoc nu nht, i khi xut

hin cc bo t mu nu (m mu

20/60/30/10).

Delicate 100-175

Bo t hnh cu hoc elip, mu nu

nht hoc nu vng (m mu t

40/80/70/10). khng bt mu khi

nhum Melzer

32

Dilatata 125-200

Phn ln bo t c dng hnh cu hoc

gn hnh cu, i khi c dng trn.

Bo t c mu m, (m mu t

60/80/20/00 n 40/60/100/00),

Lacunose.

120-175

Bo t c dng hnh cu hoc gn hnh

cu, i khi c dng elip, mu vng

(m mu t 20/60/60/00 n

20/60/100/10). Thnh bo t c cu

trc 5 lp, chia thnh 3 nhm.

Myriocarpa 150-200

Bo t c dng hnh trng hoc hnh

trn, b mt c vt lm,mu nu hoc

c mu nu nht (40/80/60/00). Thnh

bo t c 1 3 lp, bt mu vng khi

nhum Melzer.

scrobiculata 120-175

Bo t c dng hnh cu hoc elip, c

bit trn thnh t bo c qu bo t.

M mu bo t t 40/60/40/00 n

40/60/80/00. Thnh bo t c 2 lp, c

phn ng khi nhum Melzer.

33

Glomus

Aggregatum 120-175

Bo t c hnh cu hoc elip, c bit

bo t c cung bo t. Bo t thng

c mu nu m, c th xut hin

vng. Cu trc thnh r rt, gm 4 lp,

chia thnh 3 nhm.

Ambisporum 100-150

Bo t hnh cu, gn hnh cu hoc

hnh trng, a s mu nu hoc c mu

nu , (m mu t 20/60/40/00),

thnh bo t rt dy, c khong 5 6

lp.

Marcaropus 100-125

Bo t c hnh cu hoc gn hnh cu,

hnh elip. a s bo t c mu vng

hoc vng nht, (m mu t

20/40/100/10). Thnh bo t dy,

nhiu lp, bt mu vng nht trong

phn ng nhum Melzer.

34

Sclerocystis

.

Coccogena

120-150

Bo t c dng hnh cu, gn hnh cu,

c mu nu hoc nu nht

(40/80/30/10). Thnh bo t gm 2 - 3

lp.

Coremioides 100-150

Bo t c hnh trn, mu nu, i khi

c mu , (m mu t 40/60/80/00 ).

Thnh bo t c 2 lp, lp ngoi dy

v c mu nu nht n nu m.

Glomites Rhyniensis 150-200

Bo t hnh cu hoc hnh elip, c

bit trn thnh bo t c cung bo t

c mu nu m. Bo t c mu nu

m. Thnh bo t c cu trc kiu

lin tip, gm nhiu lp.

35

Entrophospora

colombiana 150-200

Bo t hnh cu, gn cu, b mt c

cc li a gic, a s c mu nu ,

s t c mu nu thnh bo t dy, c

nhiu qu bo t.

schenckii 150 -200

Bo t hnh cu, gn hnh cu hoc

hnh thn, b mt c cc gai nh. Bo

t c mu xm xn hoc nu ti

(60/80/70/10). Thnh bo t gm 2 lp

Gigaspora

Candida, 120160

Bo t thng c hnh cu v gn hnh

cu, mt s c hnh elip. Bo t c

mu nu hoc (m mu t

20/60/20/00). Thnh bo t c cu trc

ni tip, gm 3 5 lp

Albida 120-150

Bo t hnh cu hoc gn hnh cu, a

s c mu trng, vng hoc vng kem,

(m mu 60/80/60/00). Thnh bo t

c 3 lp.

36

3.1.2 Thnh phn loi AMF trong t trng cam qu hp thuc x

Minh Tn - huyn Qu Hp - Tnh Ngh An.

16 kiu hnh AMF xc nh c trong t trng cam ph qu x Minh

Tn huyn Qu Hp - tnh Ngh An thuc 6 chi, 3 h v 2 b ph.

Cc chi AMF xut hin trong t trng cam Ph Qu x Minh Tn-

huyn Qu Hp tnh Ngh An l: Acaulospora, Entrophospora, Glomus,

Sclerocystis, Glomites v Gigaspora.

Cc h c tm thy y l: Acaulosporaceae, Glomaceae v

Gigasporaceae.

Cc b ph tm thy l: Glomineae v Gigasporinae

Thnh phn loi chi tit ca 6 chi AMF trong t trng cam.

* Chi Acaulospora: C 6 loi, bao gm cc loi Acaulospora

appendiculata, Acaulospora delicate, Acaulospora dilatata, Acaulospora

myriocarpa, Acaulospora bireticulata, Acaulospora lacunose.

* Chi Entrophospora: C 2 loi, bao gm cc loi Entrophospora

colombiana, Entrophospora schenckii.

* Chi Glomus: C 3 loi l Glomus aggretum, Glomus ambisporum,

Glomus marcaropus.

* Chi Sclerocystis: C 2 loi l Sclerocystis coccogena v Sclerocystis

coremioides.

* Chi Glomites: C 1 loi duy nht Glomites rhyniensis.

* Chi Gigaspora: C 2 loi Gigaspora candida, Gigaspora albida.

3.1.3. c im phn b AMF trong t trng cam Qu Hp Ngh An

Cam c th sinh trng v cho thu hoch bnh thng t xp. Nhng

ch t tt, cy cam mi cho nng sut cao, cht lng. Vit Nam, cy

cam c trng trn 6 loi t chnh trong t bazan chim t 50 60%

din tch t trng cam.

37

3.2. PHN B CA AMF THEO GING CAM

Cc kt qu iu tra ch r rng Ph Qu, Ngh An c rt nhiu ging cam

khc nhau nhng ph bin nht l: cam X oi, Vn Du, v V2. S phn b

ca nm Mycorrihza gia cac ging cam co s khac nhau (Hnh 3.9).

Ging cam X oi

ging cam X oi pht hin c 16 loi AMF trong c:

* 6 loi thuc chi Acaulospora l Acaulospora appendiculata,

Acaulospora dilatata, Acaulospora bireticulata, Acaulospora lacunose,

Acaulospora scrobiculata, Acaulospora elegans, Acaulospora rehmii.

* 2 loi thuc chi Entrophospora l Entrophospora schenckii,

Entrophospora infrequens.

* 3 loi thuc chi Glomus l Glomus aggretum, Glomus ambisporum,

Glomus australe,

* 2 loi thuc chi Sclerocystis l Sclerocystis coccogena v Sclerocystis

coremioides.

* 1 loi thuc chi Glomites l Glomites rhyniensis.

* 2 loi thuc chi Gigaspora l Gigaspora candida, Gigaspora albida.

Ging Vn Du

Trong t trng cam Vn Du c tng s 9 loi AMF xut hin, gm:

* 4 loi thuc chi Acaulospora l Acaulospora delicate, Acaulospora

dilatata, Acaulospora myriocarpa, Acaulospora bireticulata, Acaulospora

lacunose.

* 2 loi thuc chi Entrophospora l Entrophospora schenckii,


* 1 loi thuc chi Glomus l:Glomus ambisporum, Glomus aggretum.

* 1 loi thuc chi Gigaspora l Gigaspora candida.

38

Ging cam V2

ging cam V2 pht hin c 12 loi AMF trong c:


dilatata, Acaulospora lacunose, Acaulospora rehmii.

* 2 loi thuc chi Entrophospora l Entrophospora colombiana,


* 3 loi thuc chi Glomus l Glomus aggretum, Glomus ambisporum,

Glomus marcaropus, Glomus australe.


coremioides.


Hnh 3.1. Tng s loi AMF trong cc ging cam

39

Hnh 3.2. Phn b loi AMF trong chi theo ging cam

T kt qu trn cho thy s lng loi AMF trong t trng cam ging

cam X oi l cao nht (16 loi) sau n ging cam V2 (12 loi) v thp

nht l ging cam Vn Du (9 loi) (Hnh 3.9).

Trong 6 chi AMF xut hin t trng cam Qu Hp Ngh An,

ging cam X oi c mt c 6 chi, Vn Du v V2 ch xut hin 4 chi xut

hin l Acaulospora, Entrophospora, Glomus v Gigaspora. Hai chi nm

AMF l Sclerocystis v Glomites khng xut hin t trng cam Vn Du,

chi Gigaspora khng xut hin t trng cam V2 (Hnh 3.10). Theo

Boddington v tg th nguyn nhn ca s sai khc ny c th bt ngun t

tnh cht ca ging cam [22]. n nm 2002 mt ln na Roy v tg cng

khng nh nguyn nhn sai khc cc loi l do ging cy [44].

Ging cam X oi hay cn gi l ging cam Vinh l ging a

phng hn hp, nhiu bin d, mc khe, b r ln, tn xe rng mc tt

trn c t xu. y c th c coi l ging thc vt bn a.

40

Ging cam V2 l ging nhp ni, ngun gc t Nht Bn, hnh thi cy to

trung bnh, tn ng, mt cnh hi tha ng knh thn cy to, phn cnh dy,

b khung tn khe, cy sinh trng kh, khi trng cy c t l sng cao.

3.3. PHN B CA AMF THEO TNG T

Kt qu nghin cu v s phn b ca thnh phn loi AMF trong t

trng cam Ph Qu - Ngh An theo 3 tng phu din (hnh 3.11) nh sau:

Tng 0 20 cm

Trong tng t ny c tng s 16 loi AMF xut hin bao gm:

* 6 loi thuc chi Acaulospora l Acaulospora appendiculata,

Acaulospora delicate, Acaulospora dilatata, Acaulospora bireticulata,

Acaulospora scrobiculata, Acaulospora elegans v Acaulospora rehmii.


Entrophospora schenckii v Entrophospora infrequens.

* 3 loi thuc chi Glomus l Glomus ambisporum, Glomus marcaropus

v Glomus mosseae.


coremioides.


* 2 loi chi Gigaspora l Gigaspora albida , Gigaspora candida.

Tng 20 40 cm

Khng nhiu nh tng 0 - 20cm, trong tng 20 - 40cm, ch c 12 loi

AMF xut hin, bao gm:


dilatata, Acaulospora scrobiculata v Acaulospora elegans.



* 2 loi thuc chi Glomus l Glomus aggretum, Glomus australe.

41

* 1 loi thuc chi Sclerocystis l Sclerocystis coccogena


* 2 loi chi Gigaspora l Gigaspora candida, Gigaspora albid

Tng 40 60 cm

C 9 loi AMF xut hin trong tng t ny l:

* 3 loi thuc chi Acaulospora l Acaulospora dilatata, Acaulospora

lacunose v Acaulospora elegans.

* 2 loi thuc chi Glomus l Glomus aggretum v Glomus australe.

* 1 loi thuc chi Sclerocystis l Sclerocystis coccogena.


* 2 loi thuc chi Gigaspora l Gigaspora candida v Gigaspora

decipiniens.

Hnh 3.3. Tng s loi AMF theo tng phu din

42

Hnh 3.4. Phn b loi AMF trong chi theo tng phu din

Nh vy, thnh phn loi AMF tp trung ch yu tng mt (16 loi), gim

dn xung tng 20 40 (12 loi), thp nht tng 40 60cm (9 loi). Ni

mt cch khc s lng loi nm AMF gim dn theo chiu su phu din

t trng cam. Kt qu phn b ny tng t nh phn b ca vi sinh vt ni

chung trong t ca Tortora (2002) [57].

Mt s nghin cu cho bit cam c 2 loi r l r ht v r dn. R cam

c ng knh 1mm, mu nu hoc nu . tng t mt lng r ht tp trung n

60%. C th chnh v nguyn nhn ny khin cho s lng loi AMF tp

trung ch yu tng 0 20 cm theo Newman [42]. Nhn li cc kt qu ca

cc tc gi khc trn th gii ta cng thy chiu hng tng t. Kt qu

nghin cu ca Redhead (1980) cho thy AMF gim ng k khi su tng

43

t t di 15 cm. Tuy nhin c nghin cu cng cho thy AMF c th xut

hin di cc tng phu din su hn [46]. Nm 1983, Ted v Chiristopher

pht hin thy rng t l phn trm r b AMF xm nhim cng nh trng thi

xm nhim r gim mnh khi tng v su ca phu din cc loi c [55].

3.4. XC NH KH NNG CNG SINH AMF TRN CY CAM

xc nh kh nng cng sinh ca AMF trn cy cam. Sau khi phn

lp bo t t t trng cam, chng ti tin hnh lm th nghim trn cy cam

con ny mm t ht.

Sau khi ht cam ny mm, ln 1 b sung 10-15 bo t AMF vo bu

cha cy k ch. Sau khi b sung AMF t 15 -20 ngy chng ti tin hnh

chiu di, s lng r cam trn hai l th nghim v cho kt qu (bng 3.1).

Kt qu cho thy l th nghim b sung Mycorrhiza c kh nng sinh trng

v pht trin tt hn trn l i chng khng b sung Mycorrhiza (Hnh 3.5).

Hnh 3.5. B sung AMF ln 1 (nh tri), th nghim i chng

(nh phi)

44

Ln 2 tin hnh th nghim lp li ging nh ln 1 v cho kt qu kh

quan (bng 3.2).

Hnh 3.6. Kt qu nhim AMF trn cy cam con.

Trong qu trnh lm th nghim, chng ti nhn thy vai tr ca AMF i vi

cy cam con l rt ln nh: lm tng kh nng hp th cc cht dinh dng,

mui khong v nc cho cy cam trn mi trng t ct (mi trng ngho

dinh dng) qua thi gian 3 thng cy vn pht trin bnh thng. Qua bng

thng k th chng ti nhn thy nhng cy cam con khi c b sung AMF

c kh nng sinh trng v pht trin hn nhiu so vi cy i chng: V

chiu di ca cy, cng nh chiu di v s lng r trn cy, c th hin

qua (hnh 3.5; 3.6; 3.7).

45

Hnh 3.7. Kt qu sinh trng ca cam sau 3 thng b sung AMF

S liu thu c sau cc ln o c tnh ton v x l qua phn mm

Ecxel, sai lch c tch theo cng thc:

Trong n s cy (n = 20)

Bng 3.2. Kt qu th nghim trn cy cam con

S ln o B sung AMF Th nghim C

Chiu di r

(cm)

S lng

r (ci)

Chiu di r

(cm)

S lng r

(ci)

Ln 1 3,2 0,17 3,0 0,1 2,1 0,03 2,0 0,01

Ln 2 6,7 0,17 5,0 0,1 3,8 0,01 3,0 0,05

Ln 3 13,0 0,02 6,0 0,05 4,8 0,13 4,0 0,02

Sau thi gian 3 thng chng ti tin hnh thu mu r c b sung AMF

xc nh kh nng cng sinh ca AMF trn r cam bng phng php

nhum trypan blue (0,005%) c ci tin ca Brundrett Mark v tg, (1996)

46

[24]. Sau khi xc nh kh nng cng sinh trn r cam, chng ti tin hnh thu

mu r vi s lng ln cho qu trnh tch DNA tng s.

Hnh 3.8. Kt qu nhum Tryphan blue AMF r cam:

1- Bo t AMF

2- si nm AMF

3- thnh t bo thc vt.

3.5. NHN ON GEN 18S rRNA

3.5.1. Tch chit DNA tng s

Tch chit DNA tng s l bc khi u tin hnh th nghim sinh

hc phn t. Thng thng, xc nh tinh sch DNA tng s c

chng ti tin hnh in di kim tra trn gel agarose 0,8% v o quang ph

ti hai bc sng 260nm v 280 nm. Tuy nhin qu trnh tch chit DNA tng

s t 2 ngun bo t trc tip v r cy nhim bo t thu c rt thp nn

chng ti khng tin hnh chy in di cng nh o quang ph. Theo cc

nghin cu trc th qu trnh nhn on gen 18S rRNA cng tin hnh

nhn trc tip t sn phm tch DNA tng s [36].

3.5.2. Nhn on gen 18S rRNA

Sau khi tch chit DNA tng s, chng ti s dng dch tch chit lm

khun dng trong phn ng PCR. Phn ng PCR c s dng 2 mi

47

VANS1_5 v NS21 vi nng mi thch hp l 10 nmol.

i vi phn ng PCR, vic xy dng mt chu trnh nhit ph hp l

yu t quan trng nht, bi v yu t nhit m bo cho s gin xon ca

phn t DNA, cng nh vic gn mi v ko di chui. Chu trnh nhit ti u

cho phn ng PCR trnh by trn.

Ngoi ra mt yu t khc cng khng km phn quan trng l nhit

gn mi. Nhit gn mi ty thuc vo tng loi mi c th c tnh ton

da trn nhit nng chy (Tm) ca on mi:

Tm = 20C x (A + T) + 4

0C x (G +C) + 3,3

0C

A, T, G, C: cc baz nit

Sau khi tnh c nhit Tm theo l thuyt, phn ng PCR c

tin hnh chy ti cc nhit gn mi khc nhau v chn c nhit

gn ti u l 500C trong thi gian 1 pht.

Sn phm ca phn ng PCR c tin hnh kim tra trn gel agarose

0,8% (Hnh 3.26). Kt qu chng ti nhn thnh cng on gen SSU c

kch thc khong 0,5 kb ng theo tnh ton l thuyt. Kt qu ny hon

ton ph hp vi nghin cu trc [36].

48

Hnh 3.9. Kt qu in di kim tra sn phm PCR

M: Marker thang DNA chun 1 kb.

1: DNA nhn bi cp mi SSU c kch thc 0.5 kb.

3.5.3. Tinh sch sn phm PCR

Nh ni trn, sn phm PCR sau phn ng vn cn ln mi d li

do c lng DNA cho vic gii trnh t gen, sn phm PCR c

chng ti tin hnh in di lng ln vi tng th tch mi ging l 50 l, sau

ct nhng bng DNA c hiu di tia UV trc khi thc hin tinh sch

thu DNA tinh khit nht. Kt qu in di thi gel (hnh 3.9.A).

A B

Hnh 3.10. Kt qu sn phm PCR.

M: Marker DNA chun 1 kb.

1: DNA nhn bi cp mi SSU c kch thc 0,5kb

(Hnh A thi gel sn phm PCR, Hnh B tinh sch sn phm PCR).

49

Trong qu trnh tin hnh, thun tin cho vic in di sn phm

PCR th tch ln, ba ging in di c ghp vo lm mt (Hnh 3.10. A).

Trn hnh nh kt qu, cc bng DNA u rt c hiu, r nt, khng c du

hiu b t gy v kch thc cc bng ph hp vi kch thc thit k mi

nm trong khong 0,5 kb trong khi mi d hon ton nm pha di. Do ,

on gel cha cc bng DNA c ct di tia UV sau tin hnh tinh

sch, to nguyn liu cho qu trnh gii t gen. Kt qu in di DNA sau khi

tinh sch th hin (hnh 3.10. B). Trn hnh nh in di, cc bng DNA hin

r nt, khng c du hiu t gy, kch thc ph hp v c bit l khng

cn d li mi pha di. iu chng t DNA lc ny hon ton tinh

khit, iu kin tin hnh gii trnh t gen.

50

KT LUN V KIN NGH

Kt lun

1. Trong 60 mu c kho st ti t trng cam ti x Minh Tn huyn Qu

Hp, Tnh Ngh An , nm 2011 c tng s 16 kiu hnh bo t AMF c

tm thy thuc 6 chi: Acaulospora, Entrophospora, Glomus, Sclerocystis,

Glomites v Gigaspora; 3 h: Acaulosporaceae, Glomaceae v

Gigasporaceae v 2 b ph: Glomineae v Gigasporinae.

S lng v thnh phn loi AMF trong t trng cam Qu

Hp Ngh An thay i theo ging cam, tng t.

Thay i theo ging cam: S lng, thnh phn loi AMF cao nht

vi ging cam X oi ( 16 AMF), Vn Du (9 AMF), V2( 12

AMF).

Thay i theo su tng t: Phu din 0 20 cm c s lng

loi v thnh phn a dng nht (16 loi), k l tng 20 40 cm

(12 loi) v thp nht l tng 40 60 cm (ch c 9 loi).

2. Cy cam con c kh nng sinh trng v pht trin v chiu di thn ,r v

s lng r khi nhim nm Mycorrhiza so vi cy i chng.

3. Ln u tin Vit Nam tch chit DNA t bo t Mycorrhiza trong t

v r cam. nhn c on gen 18S rRNA c kch thc khong 0,5 kb t

dch chit bo t nm v r cy nhim Mycorrhiza.

Kin ngh

* Tip tc nghin cu phn tch gii trnh t on gen 18S rRNA nh

danh chnh xc tn loi AMF.

* Tip tc nghin cu hng thu sinh khi bo t Mycorrhiza to phn bn

cho cy cam.

51

TI LIU THAM KHO

Ting Vit

1. Nguyn S Giao (1976). nh hng ca r nm i vi sinh trng v

pht trin ca cy non thng nha. T/s Lm nghip 25, s 12/1976, tr

21 - 36.

2. Nguyn S Giao, Nguyn Th Nhm (1977). Bc u nghin cu ng

dng mt s loi nm cng sinh thun chng to r nm cy con

thng nha (Pinus Merkussi). K yu khoa hc, 1976 - 1977, Vin

Khoa hc Lm nghip Vit Nam, tr 98 103.

3. Nguyn S Giao, Nguyn Th Nhm (1980). Nghin cu bnh vng ci

cy Thng nha, da vo qui lut cng sinh vi nm. K yu Khoa hc

1970 -1980. Vin Khoa hc Lm nghip Vit Nam, tr 216 224.

4. Nguyn S Giao v cng s (1998). Vin Th nhng Nng ho, Vin

Lm Nghip, Vin Cng Ngh Sinh, hi tho v nm r Mycorrhiza.

5. Nguyn Hng H, Trnh Th Thu H, Nguyn Th Hng Hi, Nguyn

Ngc Cng, Trn Ngc Ninh (2003). Thu thp bo t nm cng sinh

trong vng r quyn cy dc liu. Hi ngh Cng ngh Sinh hc ton

quc, H Ni 2003, tr 238 - 240.

6. Nguyn Vit Hip (2008) , phn b nm r cng sinh Arbuscular

mycorhiza fungi trong t trng ch Thi Nguyn. T/c Khoa hc t,

s 29, tr 17 23.

7. Trn Vn Mo s dng cn trng v vi sinh vt c ch tp 2. Nxb

Nng Nghip, H Ni 2004 tr 247-251.

8. Nguyn Th Minh (2005). Phn lp v tuyn chn nm r Arbuscular

mycorhizae x l cho cy trng. T/c Khoa hc t, s 23/2005, tr 46

-51.

52

9. Nguyn Th Minh (2007). nh hng ca mt s loi phn hu c n

s thit lp mi quan h cng sinh ca nm r Arbuscular mycorhizae,

Gigaspora margirita v s sinh trng ca cy ch. T/c Khoa hc t,

s 28, tr 27 - 31.

10. Nguyn nh Quyn vai tr ca Mycorrhiza trong nn Nng Lm

nghip hin i, hi tho v nm r Mycorrhiza. Vin Th Nhng

Nng Ha, 11/2004.

11. Nguyn Vn Sc, Bi Quang Xun, Nguyn Vit Hip (2004), nghin

cu, p dng k thut pht trin cng sinh Mycorrhiza cho mt s cy

trng chnh ti mt s vng sinh thi phc v cho sn xut nng nghip

bn vng, bo co kt qu nghin cu 2004. Vin Th Nhng Nng

Ha, H Ni 12/2004.

12. Nguyn Vn Sc, Bi Quang Xun, Nguyn Vit Hip, Nguyn Th

Nga (2005). Nm r ni cng sinh (VAM) v qun th vi sinh vt trong

t trng bi c sn oan Hng Ph Th. T/c Khoa hc t, s

23, tr 42-46.

13. Nguyn Vn Sc, Bi Quang Xun, Nguyn Vit Hip (2006). Kh

nng nhn bo t nh cc cy k ch ca 3 chng nm r ni cng

sinh (Vesicular Arbuscular mycorhiza) SHM 4-DH16, SHM 04-DH47

v SHM 04-TC 139 phn lp t t Vit Nam. T/c Khoa hc t, s 24,

tr 33 - 37.

14. Phm Quang Thu v Nguyn c Thng (1998). Bc u nghin cu

thnh phn loi nm ngoi cng sinh vi mt s loi Thng Vit

Nam, Thng tin khoa hc Lm nghip 1.

15. Phm Quang Thu (1999). ng dng vi sinh vt cng sinh trong vic

sn xut cy con vn m. Tp ch Khoa hc, Cng ngh v Qun l

Kinh t, Nng nghip v Cng nghip Thc phm.

53

16. Phm Quang Thu (2002) - S dng nm cng sinh v vi sinh vt phn

gii pht pht sn xut cy con Thng nha cht lng cao vn

m. Bn tin trng mi 5 triu ha rng.

17. Nguyn Hong Yn (2007). Phn b nm ni cng sinh vi loi Sao

en (Hoperata Odorata Roxb) ti tnh ng Nai. Lun vn Thc s

Khoa hc Lm nghip, Trng i hc Lm nghip.

Ting nc ngoi

18. Abbott L. K, Robson A. D. (1982). Infectivity of vesicular Arbuscular

Mycorrhiza Fungi in agricultural soils. Aust J. Agric Res. 33, pp. 1049

1059.

19. Bagyaraj D. J. (1984). Biological interactions with VA mycorrhizal

fungi. In: VA mycorrhizae. Bailey C. L. and Mansfield J. W. CRC

Press, Boca Raton, Florida, USA, pp.131 - 154.

20. Bali V, Mammta J, and Mukerji T. (2002). Effect of VAM fungi on

Fusarium wilt of cotton and jute. ACOM 1, University of Madras, p. 2.

21. Barea J. (1982). Production of plant growth regulating substances by

VAM fungus Glomus mosseae. Applied and Environmental

Microbiology. Vol 43, No 3, pp. 810 - 813.

22. Boddington C. L, Dodd, J. C. (2000). The effect of agricultural

practices on the development of indigenous Arbuscular Mycorrhiza

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59

PH LC

Hnh 2.3. Thu mu t v r cam ti Qu Hp - Ngh An

Hnh 2.4. Sng v lc t qua ry

Hnh 2.5. Ly tm dch AMF(nh tri) v lc ht chn khng mu (nh phi)

60

Hnh 2.6. Soi mu, pht hin bo t AMF nh knh hin vi

Hnh 2.7. Chp nh bo t AMF qua knh hin vi

Hnh 2.8. m bo t qua (nh tri) v gi ging bo t (nh phi)

i

LI CM N

Ti xin by t lng bit n su sc ti TS. Nguyn Huy Hong

Trng Phng Vi sinh vt t Vin Cng ngh sinh hc, tn tnh hng

dn, to mi iu kin thun li gip v chia s nhng kh khn cng ti

trong qu trnh lm vic v hon thnh lun vn.

Ti xin c gi li cm n n Ths. Nguyn Vit Hip, Ph Trng

b mn vi sinh vt Vin Th nhng nng ha. gip ti trong qu

trnh lm th nghim ti phng th nghim. Qua y ti cng xin by t lng

bit n su sc n cc c, cc ch v cc em trong Phng Vi sinh vt t

gip ti rt nhiu trong thi gian thc tp ti phng.

Ti xin chn thnh cm n cc thy, cc c tham gia ging dy ti Khoa

sinh, Trng i hc khoa hc, i Hc Thi Nguyn.

Cui cng ti xin gi li tri n ti b m, ngi thn v bn b chia

s nhng kh khn trong cuc sng v cng vic ti hon thnh tt lun

vn.

Mt ln na ti xin chn thnh cm n s gip qu bu .

Thi nguyn, ngy 30 thng 7 nm 2012

Hc vin

L Th Thy

ii

LI CAM OAN

Ti xin cam oan y l cng trnh nghin cu khoa hc ca ti, cc s

liu v kt qu trong lun vn l trung thc v cha tng c ai cng b

trong bt k cng trnh nghin cu khoa hc no khc.

Thi nguyn, ngy 30 thng 7 nm 2012

Hc Vin

L Th Thu

iii

MC LC

Li cm n ................................................................................................................... i

Li cam oan .............................................................................................................. ii

Mc lc ...................................................................................................................... iii

Danh mc ch vit tt ................................................................................................ v

Danh mc bng trong lun vn .................................................................................. vi

Danh mc hnh trong lun vn ................................................................................ viii

M U ........................................................................................................... 1

CHNG I: TNG QUAN TI LIU ........................................................... 3

1.1. LCH S HNH THNH TN GI ARBUSCULAR MYCORRHIZA

FUNGI (AMF) .................................................................................................. 3

1.2. PHN LOI MYCORRHIZA .................................................................... 4

1.3. PHN LOI BO T AMF ..................................................................... 8

1.4. CC PHNG PHP S DNG TRONG NGHIN CU

MYCORRHIZA .................................................................................................. 9

1.5. S PHT TN BO T V NH HNG CA AMF I VI

THC VT CH ........................................................................................... 11

1.6. TNH HNH NGHIN CU AMF TRN TH GII BNG K

THUT SINH HC PHN T ..................................................................... 15

1.7. TNH HNH NGHIN CU VIT NAM ........................................... 17

CHNG II NGUYN LIU V PHNG PHP NGHIN CU ......... 20

2.1. NGUYN LIU ....................................................................................... 20

2.1.1. Mu ....................................................................................................... 20

2.1.2. Ha cht v thit b nghin cu ............................................................ 20

2.2. PHNG PHP NGHIN CU............................................................ 21

2.2.1. Phng php nghin cu ....................................................................... 21

2.2.1. Phng php tch bo t AMF.21

iv

2.2.2. Tch DNA tng s25

2.2.3. in di DNA trn gel agarose..25

2.2.4. K thut PCR27

2.2.5. Tinh sch sn phm PCR..29

CHNG III KT QU V THO LUN.31

3.1. PHN LOI AMF TRONG CC MU NGHIN CU.31

3.1.1. Hnh thi v cu to bo t AMF trong t trng cam Qu Hp -

Ngh An..31

3.1.2. Thnh phn loi AMF trong t trng cam Ph Qu thuc x Minh

Tn- huyn Qu Hp - tnh Ngh An ............................................................. 36

3.1.3. c im phn b AMF trong t trng cam Qu Hp Ngh An..... 36

3.2. PHN B CA AMF THEO GING CAM ......................................... 37

3.3. PHN B CA AMF THEO TNG T ............................................. 40

3.4. XC NH KH NNG CNG SINH AMF TRN CY CAM ......... 43

3.5. NHN ON GEN 18S rRNA .............................................................. 46

3.5.1. Tch chit DNA tng s ........................................................................ 46

3.5.2. Nhn on gen 18S rRNA .................................................................... 47

3.5.3. Tinh sch sn phm PCR ...................................................................... 48

KT LUN V KIN NGH ........................................................................ 50

Kt lun ........................................................................................................... 50

Kin ngh ......................................................................................................... 50

TI LIU THAM KHO ............................................................................... 51

PH LC ...59

v

DANH MC CC T VIT TT

A : Arbuscular

AMF : Arbuscular Mycorrhiza Fungi

DNA : Axit Deoxyribonucleic

dNTP : deoxynucleoside triphosphate

ECM : Ectomcorrhizas

Kb : Kilobase

PCR : Polymerase Chain Reaction

RADP : Radom Amplified Polymorphism DNA (Phn tch

ADN a hnh c nhn bn ngu nhin)

RFLP : Restriciton Fragment Length Polymorphis (Phn

tch chiu di cc on DNA ct hn ch)

V : Vesicules

VAM : Vesiculer Arbuscular Mycorrhiza

vi

DANH MC HNH TRONG LUN VN

Hnh 1.1: S cng sinh ca AMF trong r cy trng. ....................................... 4

Hnh 1.2: Biu cy pht sinh chng loi AMF ............................................ 7

Hnh 2.1: Bng mu so snh phn loi nm Mycorrhiza ........................... 24

Hnh 2.2: Chu trnh nhit PCR ........................................................................ 29

Hnh 3.1: Tng s loi AMF trong cc ging cam ......................................... 38

Hnh 3.2: Phn b loi AMF trong chi theo ging cam .................................. 39

Hnh 3.3: Tng s loi AMF theo tng phu din........................................... 41

Hnh 3.4: Phn b loi AMF trong chi theo tng phu din ........................... 42

Hnh 3.5: B sung AMF ln 1 (nh tri), th nghim i chng (nh phi) ... 43

Hnh 3.6: Kt qu nhim AMF . ...................................................................... 44

Hnh 3.7: Kt qu sinh trng ca cam sau 3 thng b sung AMF ................ 45

Hnh 3.8: Kt qu nhum Tryphan blue AMF ............................................... 46

Hnh 3.9: in di kim tra sn phm PCR . .................................................... 48

Hnh 3.10: in di thi gel sn phm PCR. . .................................................. 48

vii

DANH MC BNG TRONG LUN VN

Bng 1.1: c im nhn dng ca mt s loi bo t AMF ........................... 5

Bng 1.2. Kt qu nghin cu AMF bng sinh hc phn t [45] ................... 16

Bng 2.1: Cp mi dng cho phn ng PCR .................................................. 20

Bng 2.2 Thnh phn phn ng PCR .............................................................. 28

Bng 3.1 kt qu phn loi bo tvAMF..31

Bng 3.2. Kt qu th nghim trn cy cam con ............................................. 45

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