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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
http://www.lrc-tnu.edu.vn
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
http://www.lrc-tnu.edu.vn
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
http://www.lrc-tnu.edu.vn
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
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http://www.lrc-tnu.edu.vn
Tn loi
Hnh dng
Ambisporum
Marcaropus
Appendicul
a
Delicate
Dilatata
Gigaspora
100-150
100-125
Thnh bo t mng.
Thnh bo t mng, c qu bo t.
Hnh cu
C mu vng nht
150-175
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
150-200
C mu nu hoc nu nht
120-175
Thnh bo t mng,1-2 lp
colombiana
schenckii.
Coccogena
C mu nu nht
100-150
Coremioides
Bo t c hnh cu,
C mu nu, nu m
120-180
Rhyniensis
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
C mu vng nht
120-150
Sclerocystis
Glomites
120-175
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
Mu sc
Kch
thc
(m)
Thnh bo t c 3 lp chia lm 2
nhm
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
15
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
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
60
74
110
Glomeromycota (except
Archaeosporaceae)
44
Cc nhm chnh vi
Glomeromycota
44
18
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
21
23
24
Bc 2:
Bc 5:
Bc 6:
Bc 7:
pht.
Bc 9:
Bc 10:
25
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
1U
0,25
DNA khun
Tng
25
28
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
100-175
Acaulospora
Delicate
31
Dilatata
Lacunose.
Myriocarpa
scrobiculata
125-200
120-175
150-200
120-175
32
Aggregatum
120-175
Glomus
Ambisporum
Marcaropus
100-150
100-125
33
Coccogena
120-150
100-150
150-200
Sclerocystis
.
Coremioides
Glomites
Rhyniensis
34
colombiana
150-200
Entrophospora
schenckii
Candida,
150 -200
120160
Gigaspora
Albida
120-150
35
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.
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
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
43
44
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.
47
48
49
Gigaspora;
h:
Acaulosporaceae,
Glomaceae
2.
3.
4.
5.
6.
7.
8.
51
9.
10.
11.
12.
13.
14.
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.
52
16.
17.
Ting nc ngoi
18.
19.
20.
21.
fungus
Glomus
mosseae.
Applied
and
Environmental
23.
24.
53
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
endophyte.
Transactions
of
the
British
42.
43.
44.
45.
46.
47.
48.
49.
Shipra Singh, Anita Pandey, Bhaskar Chaurasia and Lok Man S. Palni
(2008). Diversity of Arbuscular Mycorrhiza Fungi associated with the
rhizosphere of tea growing in natural and cultivated ecosites.
Canadian Journal of Microbiology, 1993, 39(6): pp 567-575.
50.
51.
52.
53.
54.
55.
56.
57.
/Cummings
Publishing
Company,
810pages
with
illustrations.
58.
59.
Vancura
V,
Kunc
F.
(1989).
Interrelationships
between
57
61.
62.
http://www.ffp.csiro.au/research/mycorrhiza/
63.
http://www.invam.caf.wvu.edu/
64.
http://www.mycorrhiza.ag.utk.edu/
58
PH LC
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
iv
DANH MC CC T VIT TT
A
: Arbuscular
AMF
DNA
: Axit Deoxyribonucleic
dNTP
: deoxynucleoside triphosphate
ECM
: Ectomcorrhizas
Kb
: Kilobase
PCR
RADP
: Vesicules
VAM
vi
vii