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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

Ambisporum
Marcaropus
Appendicul
a
Delicate
Dilatata

Gigaspora

Thnh bo t mng, c 2 lp.

100-150
100-125

Thnh bo t mng.
Thnh bo t mng, c qu bo t.

Hnh cu

C mu vng nht

150-175

Thnh bo t dy, 1-6 m.

C mu nu, nu m
C mu nu, en

100-175
125-200

Thnh bo t dy.
Thnh bo t dy,c nhiu lp.

C mu nu hoc nu nht

120-175

Thnh bo t dy, c vch ngn.

C mu nu nht hoc vng nht

150-200

Thnh bo t dy, c 3-4 lp.

C mu nu hoc nu nht

120-175

Thnh bo t mng,1-2 lp

colombiana

Hnh cu hoc hnh trng

schenckii.

Hnh cu ,hnh trn

C mu nu nht hoc mu vng

Coccogena

Bo t c dng hnh cu, gn hnh


cu, elip

C mu nu nht

100-150

Coremioides

Bo t c hnh cu,

C mu nu, nu m

120-180

Rhyniensis

Hnh cu hoc hnh elip

C mu nu m

150-200

Candida,

Bo t thng c hnh cu v gn
hnh cu, mt s c hnh elip

C mu nu, nu m, mu nu

120160

Albida

Bo t c dng hnh cu, hoc elip

C mu vng nht

120-150

Sclerocystis

Glomites

120-175

Hnh cu hoc hnh trng


Hnh cu hoc hnh trng

c qu bo t, hnh cu
Hnh cu, c qu bo t
Acaulospora
C qu bo t, hnh cu hoc gn
Myriocarpa,
hnh cu.
Bireticulata
Hnh cu, dng qu l
Lacunose.
Hnh cu

Entrophora

c im

C mu nu, mu nu , mt s c
mu vng nht
Mu nu, nu m
Nu hoc mu vng nht

Aggregatum Hnh cu, hnh trng, c cung nh


Glomus

Mu sc

Kch
thc
(m)

C mu nu nht hoc mu vng nht 120-200

Thnh bo t c 3 lp chia lm 2
nhm

150-200 Thnh dy chia bo t thnh 3 lp.


Thnh bo t mng c 2 lp.
Thnh bo t c 3 lp,chia lm 2
nhm.
Thnh bo t c cu trc kiu lin
tip, gm nhiu lp.
Thnh bo t c 3 lp chia lm 2
nhm
Thnh bo t c cu trc ni tip,
gm 3 5 lp.

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].

Hnh 1.2. Biu cy pht sinh chng loi AMF


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

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).

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].

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 90 oC 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].
10

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
11

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
12

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]:
13

* 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.
14

- 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.

15

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


Vng
gen

SSU
rDNA
Genomic
DNA

K thut sinh
hc phn t

on mi

VANS1

PCR PCR
RADP

T bo ch

Ti liu
tham kho

Glomales

109

Glomusversiforme
OPA-02 v OPA-04 Gl. mosseae . Gl. Caledoniu
OPA-18 v P124
Acaulosporalaevis
Gigasporamargarita
OPA-18 v P124
Scutellospora gregaria

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

SS38 v VANS1

R v bo t ca AM,

VANS1

Scutellospora v Glomus

VAGIGA

Gigasporaceae

Partial
rDNA

PCRpartial

30

ITS1 and
ITS2

PCR

ITS1 v ITS2

G. margarita

32

ITS

PCR

ITS1 v ITS4

G.mosseae v Gigaspora
margarita

29

LR1 v FLR2

Di nhm ca Glomales

FLR2-5.23 v
FLR2-8.23

G. mosseae, G. intraradices

SSU
rDNA

PCR-RFLP
PCRnested

45

G. roseae

LR1-23.46
28S
rDNA

PCRSSCPs

LSU-Primers

Glomus sp.

82

SSU
rDNA

PCR

NS31 v AM1

Glomus sp.

16

Di nhm ca Glomales

SSU
rDNA

PCRSSCP

VANS1

Nested-PCR

ITS, AM1

ITS

PCRRFLP

ITS1 v ITS4

G. mosseae

ITS

Nested PCR
SSCP

Eukaryotic
universal primer

Glomus sp.

ITS

Nested-PCR

ITS 5 v ITS4

PCR

SSU-Glom/LSUGlom 1

ITS

ITS5 v ITS4

Glomus-specific ITS primer

60
74
110

Glomeromycota (except
Archaeosporaceae)

44

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
17

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.

18

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.
19

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
VANS1_5
NS21

Trnh t nucleotide
5' GTC TAG TAT AAT CGTTAT ACA GG 3'
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.
20

- 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.

21

+ 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].
22

- 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.

23

Hnh 2.1. Bng mu so snh phn loi nm Mycorrhiza

24

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

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

pht.

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

25

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.
26

* 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.
27

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

Nc kh ion v trng

Th tch (l)
11,75

Buffer

1X

2,5

dNTP

10 mM

0,5

Mi xui

10 pmol

Mi ngc

10 pmol

Taq DNA polymerase

1U

0,25

DNA khun

Tng

25

28

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 95 0C 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.
29

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.

30

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
c im
Hnh dng
thc m

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).

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

Acaulospora

Delicate

31

Dilatata

Lacunose.

Myriocarpa

scrobiculata

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),

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.

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.

120-175

32

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.

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.

Glomus

Ambisporum

Marcaropus

100-150

100-125

33

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.
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.

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.

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.

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.

Sclerocystis
.

Coremioides

Glomites

Rhyniensis

34

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.

Entrophospora

schenckii

Candida,

150 -200

120160

Gigaspora

Albida

120-150

35

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

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
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.

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 Tnhuyn 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.
36

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,
Entrophospora infrequens.
* 1 loi thuc chi Glomus l:Glomus ambisporum, Glomus aggretum.
* 1 loi thuc chi Gigaspora l Gigaspora candida.

37

Ging cam V2
ging cam V2 pht hin c 12 loi AMF trong c:
* 4 loi thuc chi Acaulospora l Acaulospora delicate, Acaulospora
dilatata, Acaulospora lacunose, Acaulospora rehmii.
* 2 loi thuc chi Entrophospora l Entrophospora colombiana,
Entrophospora infrequens.
* 3 loi thuc chi Glomus l Glomus aggretum, Glomus ambisporum,
Glomus marcaropus, Glomus australe.
* 2 loi thuc chi Sclerocystis l Sclerocystis coccogena v Sclerocystis
coremioides.
* 1 loi thuc chi Glomites l Glomites rhyniensis.

Hnh 3.1. Tng s loi AMF trong cc ging cam

38

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.
39

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.
* 2 loi thuc chi Entrophospora l Entrophospora colombiana,
Entrophospora schenckii v Entrophospora infrequens.
* 3 loi thuc chi Glomus l Glomus ambisporum, Glomus marcaropus
v Glomus mosseae.
* 2 loi thuc chi Sclerocystis l Sclerocystis coccogena v Sclerocystis
coremioides.
* 1 loi thuc chi Glomites l Glomites rhyniensis.
* 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:
* 4 loi thuc chi Acaulospora l Acaulospora delicate, Acaulospora
dilatata, Acaulospora scrobiculata v Acaulospora elegans.
* 2 loi thuc chi Entrophospora l Entrophospora colombiana,
Entrophospora infrequens.
* 2 loi thuc chi Glomus l Glomus aggretum, Glomus australe.
40

* 1 loi thuc chi Sclerocystis l Sclerocystis coccogena


* 1 loi thuc chi Glomites l Glomites rhyniensis.
* 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.
* 1 loi thuc chi Glomites l Glomites rhyniensis.
* 2 loi thuc chi Gigaspora l Gigaspora candida v Gigaspora
decipiniens.

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
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 vng ng c gi l r ht. R dn 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
42

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)

43

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).

44

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
Chiu di r S
(cm)

Ln 1

3,2 0,17

Ln 2
Ln 3

Th nghim C
lng Chiu di r S lng r
(ci)

(cm)

(ci)

3,0 0,1

2,1 0,03

2,0 0,01

6,7 0,17

5,0 0,1

3,8 0,01

3,0 0,05

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)
45

[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
46

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) + 40C x (G +C) + 3,30C
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].

47

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).

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).

48

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.

49

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

Gigaspora;

h:

Acaulosporaceae,

Glomaceae

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.
50

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.

51

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


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58

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)


59

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)

60

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

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

ii

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

iii

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

iv

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

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

vi

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

vii

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