TAP CHl KHOA HOC VA CONG NGHE Tap 48, so 1, 2010 Tr. 73-79
ANH HU'O'NG CUA HAM LU'O'NG NITO VA PHOTPHO TRONG OAT OEN KHA NANG CONG SINH CUA NAM ARBUSCULAR MYCORRHIZAS TREN C A Y NGO VA HIEU QUA XU Ll O A T O
NHIEM CHI
BUI VAN C U O N G , TANG THj CHINH
1. M O DAU
Dung thuc vat dl cai tao nhiing vung dat 6 nhiem kim loai nang (phytoremediation) la bien phap dang nhan dugc su quan tam dac biet bai nhiing im viet cua no so vai cac bien phap khac:
than thien vai moi truang, chi phi re, de ap dung va co the ap dung cho mgt vung 6 nhiem rgng Ion... Tac nhan chinh trong phuang phap nay la cac loai thuc vat co kha nang hap thu mgt lugng lan kim loai ttr dat va tich tu trong sinh khoi cua chiing dan den ham lugng kim loai con lai trong dat bi giam di [4]. Song nhiing nghien ciiu gan day lai danh gia cao vai tro cua he vi sinh vat song cpng sinh vai re thuc vat dac biet la nam arbuscular mycorrhiza (AM), tham chi tac gia Jamal (2002) con dua ra khai niem "Mycorrhizo-Remediation" de cho thay vai tro khong nho cua nam AM trong viec cai tao dat 6 nhiem [3]. Nam Arbuscular Mycorrhizas (con dugc ggi la nam ngi cgng sinh Endomycorrhiza) la mpt trong ba dang chinh cua nam cgng sinh Mycorrhizas dugc mieu ta lan dau tien vao nam 1885 bai nha thuc vat hgc nguai Diic A. B. Frank. Vai tio cua chiing trong nong nghiep va trong rimg da dugc danh gia va cong nhan qua cac nghien ciiu rat kl luang trong vong 40 nam tio lai day [7]. Song nhiing vai tro ciia nam AM trong ITnh vuc bao ve moi truang van chua dugc quan tam nhieu. Trong cac nghien ciiu gan day cac nha khoa hgc da chiing minh dugc vai tio thiet yeu cua nam cgng sinh arbuscular mycorrhizas khong nhirng trong viec giiip cay chong chiu tot trong dieu kien 6 nhiem kim loai, ma con nang cao hieu xuat hap thu kim loai nang cua cay xu li [2]. Co nhieu yeu to anh huang den qiia trinh phat trien cua thuc vat ciing nhu kha nang cpng sinh ciia nam AM vao bg re ciia cay chu nhu nhiet dp, anh sang, dp am ciia dat, thanh phan cac chat dinh duang co tiong moi truang dat. Duai day la cac ket qua nghien ciiu anh huong ciia ham lugng nito va photpho den su cgng sinh ciia nam AM vao re cay ngo va kha nang xii ly dat 6 nhiem chi cua chiing.
2. PHUONG PHAP NGHIEN ClTU 2.1. Doi tuong nghien cuu
Chung nam cgng sinh arbuscular mycorrhizas thugc chi Glomus do phong Vi sinh vat moi truang, Vien Cong nghe moi truonng phan lap va cung cap. Giong ngo dugc su dung trong nghien ciiu la giong ngo lai LVN - 4 cua Vien nghien ciiu ngo (Dan Phugng - Ha Npi). Kim loai chi (Pb) dugc bo sung duai dang muoi PbCl2 do Trung Quoc san xuat.
2.2. B6 tri thi nghiem
Thi nghiem dugc bo tri thanh dai tang dan ham lugng dinh duong P va N tiong d4t (bo sung phan hiiu co MIX 209 ciia Cong ty co phan Khanh Sinh).
TNI: Trpn 50% dit voi 50% cat (tuong iing voi P: 252,3 ppm va N: 44,1 ppm);
TN2: Trpn 80% dk voi 20% cat (tiiong iing voi P: 363,1 ppm va N: 70,6 ppm);
TN3: Tring tren dat thudng (tuong iing vai P: 603,3 ppm va N: 85,2 ppm). Cac cay thi nghiem TN3 trong qua trinh nuoi trong bi chupt pha hoai, nen cac kit qua cua TN3 chiing toi khong de cap den do khong phan anh dugc ban chat cua ket qua;
TN4: B I sung vao dat 0,3% phan hihi ca (tuong iing voi P: 718,6 ppm va N: 86,8 ppm);
TN5: B I sung vao dat 0,6%o phan hiru co (tuong iing voi P: 752,5 ppm va N: 87,6 ppm);
TN6: B I sung vao d4t 0,9%) phan hiiu co (tuong tmg voi P: 786,5 ppm va N: 88,2 ppm);
TN7: B I sung vao dat 1,2%) phan hiiu co (tuong iing voi P: 900 ppm va N: 93,8 ppm);
TN8: B I sung vao dat 1,5%) phan huu co (tuong iing voi P: 952,3 ppm va N: 105 ppm).
Ttr TNI den TN8 dugc bo sung them 2000 mg Pb (Pb dugc bo sung duai dang muoi PbCli.
Moi cong thiic dugc lap lai 3 lan. Bao tir Glomus dugc bom true tiep vao bp re cay ngo sau bon ngay nay mam, voi so lugng bao tir tii 30 - 40 bao tii cho mot cay.
2.3. Phuong phap phan tich
Phuang phdp thu thdp bdo tic AM tic ddt vimg re ciia cdy chu: phuong phap chinh dugc chiing toi sir dung trong nghien ciiu la phuang phap ray am (wet seiving): 50 - lOOg dat dugc hoa voi nuoc trong mpt chiec ca dung tich 0,5 - 1 lit khuay deu len, nhiiTig cue dat Ion dugc bop nho bang tay. Sau khi khuay deu ta de lang khoang 20 giay va do dich noi vao nhiing chiec ray da dugc chuan bi tru'oc vai cac kich thuoc 16 ray lan lugt la: 500 pm, 200 pm, 50 pm. Qua trinh nay dugc lap lai 3 lan. Sau do nhiing thanh phan con dinh lai 6 ray dugc chuyen den dia petri.
Su dung kinh hien vi xuoi chieu (disecting microscope) de quan sat va lay timg bao tir ra tir dia petri. Bao tii dugc bao quan trong nuoc cat 6 4°C [4].
Phucmg phdp nhugm mdu de quan sdt cdu true dien hinh cua AM trong re: Sir dung phuong phap nhupm mau voi dung dich thuoc nhupm fucsin axit 0,01%) trong lactoglyxerol cho phep ta quan sat dugc nhiing cau tnic dac trung ciia AM ben trong to chiic re. Sau khi da loai bo dat cat, re dugc xu ly vai dung dich KOH 10%) trong khoang 1 - 2 gib a 90°C. Sau qua trinh tay, re dugc ngam trong dung dich HCI 2% trong 10 phiit, muc dich la de trung hoa boft kiem ttong qua trinh tay, sau do dugc rira lai bang nuoc cat. Re dugc nhupm mau bang dung dich fticsin axit 0,01% trong nuoc nong voi thai gian tii 10 - 15 phiit. De lam giam b6l mau nhupm giup cho viec quan sat de dang hon, re dugc nia voi dung dich axit lactic 2 - 3 lan. Liic nay re da san sang cho viec quan sat [8].
Phuang phdp xdc dinh mat do cua AM trong re: Sii dung phuong phap line insect: Mau rl dugc lay ngau nhien tir bg ri ciia cay, loai bo dat cat, sau do dugc cat thanh nhiing mau nho khoang 1-1,5 cm, vai s l lugng la 50 mSu. Nhtmg miu re nay dugc nhugm mau, sau do dugc quan sat duoi kinh hiln vi. Mat dp nam AM trong bp r l cua cay dugc tinh nhu sau:
, , , y m a u x u a t h l e n AM _,^^ ^^^
M a t d 6 ( % ) = ^ ^ ^ •- 100 [2].
2^ mau L/dc quan sat
Xae dinh hdm luang Nita tong so trong ddt bdng phuang phdp Kendal [1 ].
Xdc dinh hdm luang Photpho tong so trong cdc mdu ddt bang phuang phdp axU ascorbic - antimoantartrat. Nguyen ly la sii dung axit pecloric hoa tan cac hgp chat photpho trong d4t. Xae dinh ham lugng photpho trong dung dich bSng phuong phap tr4c quang " mau xanh molypden"
3. KET QUA VA THAO LUAN
3.1. Kha nang sinh truong cua cac cay thi nghiem 6" nhirng cong thuc khac nhau
p^^^^ Sinh khoi tong —•— Sinh khoi r^
Hinh I.A: Sir bien dong chieu cao; B: Sinh khoi kho cila ngo sau 11 tuan thi nghiem
Qua bieu do (hinh IA) chiing ta co the thay su phat trien ve chieu cao cua cac cay thi nghiem trong dat giau dinh duong kha nhanh va tot (55 cm sau 11 tuan), trong khi cac cay a dat ngheo phat trien kem hon ban (39 cm sau 11 tu4n).
Luang sinh khoi thu dugc cua cac cay thi nghiem cho ta thay mpt sir khac biet kha ro rang giiia cac cong thiic khac nhau. Sinh khoi tong ciia cac cay thi nghiem co xu huong tang ti le thuan theo ham lugng phan bon bo sung vao dat trong, nhung trai lai sinh khoi re lai phat triln theo ti le nghich vai ham lugng phan bon bo sung vao dat (hinh IB va 2).
Qua nhiing nghien ciiu ciia minh, tac gia Andre Fortin [2] cho rang su phat tiien manh me bp re a viing dat ngheo la do su tuong tac qua lai giiia cay chu va nam cpng sinh thong qua cac tin
hieu thong tin 6 cap dp phan tir (dang phytohocmon), qua do bp ri cua cay se dugc phat triln mpt each toi da lam gia tang kha nang lien ket giura nam va bg re cua cay giiip cho he sgi n4m co the phat trien va vuon tai nhiing diem each rat xa bp re de hiit chat dinh duong.
3.2. Hinh thai va mat do nam AM trong bo re ngo
Hinh 3A la tieu ban cho thay he sgi nam AM nam trong c4u tnic ciia ri, chiing nSm xen ke va song song voi chieu sap xep cua cac te bao, co luc tao thanh cac dang bgng (vesicular) dac trung cua nkm arbuscular mycorrhizas.
w ^ :
Mat dp nam AM dat cao nhk a nhung cay thupc TNI (22,8%) va giam dan din gia tii thip nhat o TN8 (5,13%). Trong thi nghiem nay, dat giau dinh duang lai la ylu t l ngan can su cpng sinh cua nim AM vao cay ngo. 6 nhiing cong thiic dit cang ngheo dinh duong (TNI, TN2) kha nang cpng sinh ciia nam AM cang cao.
m Mat dp nam AM
1 n
_ D
B TNI TN2 TN4 TN5 TN6 TN7 ' TN8
Hinh 3. A: He sgi nam AM trong re ngo; B: Mat do nam AM trong re ngo a cac thi nghiem khac nhau
3.3. Hieu suat su dung P va N
Bangl. Hieu suat sii dung P va N cua cac cay thi nghiem Ki hieu mau
Hieu suat sir dung P (%) Hieu suat sii dung N (%)
TNI 40,8 30
TN2 34,7 22,5
TN4 29,4 19
TN5 26,3 17,5
TN6 23,8 17
TN7 22,3 16,4
TN8 22,6 16
Hinh 4. Hieu suat su dung P va N cua cay ngo
Hieu suat sii dung dugc tinh theo ham lugng P va N trong dat giam di trong qua trinh nuoi tilng. Nhin chung hieu suit hip thu P ciia cay cao hon so voi hieu suat sii dung N va giam din tir cong thiic TN 1 din TN8 (bang 1, hinh 4).
Su phat triln manh me cua bp re voi mat dp nim AM cao giiip cho viec hap thu dinh duong hieu qua hon khi cay phai sing trong dilu kien ngheo dinh duang.
3.4. Ham luong Pb tich luy trong sinh khoi
Bdng 2. Ham lugng Pb tich liiy trong sinh khoi Ki hieu mau
Than la (mg/kg) Re (mg/kg)
TNI 105,2 915,2
TN2 149 789,2
TN4 119,7 735,9
TN5 114,3 718,6
TN6 97,7 682,2
TN7 92,1 675,9
TN8 92,3 604,2
0 Ham iugng Pb trong than la
160 140 120 100
o) 40 E
20
_92,1 __92.3_
TNI TN2 TM4 TM5 TM6 TN7 TM8
0 Ham iuang Pb trong re |
1000 p 9 i 5 7 2 -
900 —fm.-
-o 700 600
^ 500 ' " 400
£O) 300 O) E 200 —
100 - 0
735.9 7 1 8 . 6 -
682.2 675.9 r ^ 604:2
g TN1 TM2 TM4 TM5 TN6 TN7 TM8
' Hinh 5. A: Ham lugng Pb tich Iriy trong than la; B: Ham lugng Pb tich liiy trong ri
Ham lugng Pb tich liiy tiong re gap hon tir 6 den 8 lan so voi than la, day la kit qua thudng xay ra tiong nhiing nghien ciiu truoc do, vi re la bg phan true tiep hap thu cac chat 6 nhiim trong dat nen thudng co mgt lugng Ion cac chat 6 nhiem tich tu lai tiong sinh khoi cua r i (bang 2).
Ham lugng Pb tich tii trong ri giam din tii TNI (915,2 ppm) din TN8 (604,2 ppm) va lugng Pb tiong re cua TNI gap 1,5 lan so voi TN8 (bang 2, hinh 5).
Mpt tiong nhiing nguyen nhan khac dan den su gia tang cac chit 6 nhiim tich tu trong bp r i d6 la nam AM, mpt lugng Pb khong nho tich tu tiong he sgi nim AM khi chiing cpng sinh trong bp ri cay chu. Dilu nay dugc chiing minh khi ta so sanh hai bilu d l hinh 3B va 5B, qua hai bilu do nay cho ta thay mat do nam va ham lugng Pb tich tu trong sinh khii r i la ti le thuan voi nhau, mat do nam cang Ion thi kha nang tich Itiy Pb cang cao va ngugc lai.
Nhu vay, viec tang cuong kha nang cgng sinh ciia nam AM khong nhiing giiip cho cay co the chong chiu tot trong dieu kien 6 nhiem kim loai, ma con lam tang kha nang hip thu chit 6 nhiem vao sinh khoi cay, dan den hieu qua xtr li cao ban.
3.5. Hieu suat xu li chi trong dat 6 nhiem ciia ngo a cac thi nghiem khac nhau
Nhin tren bieu do hinh 6 ta co the thay, hieu suat xu li cua TNI va TN2 chi dat gia tri trung binh (3% - 3,54%) mac du kha nang tich tu Pb ciia chiing trong sinh khii la cao nhit. Trai lai, cac cay thugc TN7 va TN8 kha nang tich tu Pb kem, nhung hieu suit xii li co phin tit hon TN 1 va TN2 (TN7: 3,78%; TN8: 4,15%).
Hieu suit xu li dat toi uu d cac cay thupc TN5 (4,92%). Ci ham lugng phan bon bo sung la 0,6% (tuong duong vdi ham lugng P: 752,5 ppm va N: 87,6 ppm) chiing vua tao dugc cho cay c6 su sinh trudng tot ve sinh khoi ma lai khong ngan can su cgng sinh ciia nim AM.
Do vay khi tien hanh sir dung thuc vat dl cai tao dit 6 nhilm, chiing ta khong nen qi 'U trpng vao viec phat triln sinh khoi cua cay bing viec bon nhieu phan, vi dilu nay gian tie 'im giam kha nang hip thu kim loai ciia cay xtr li do sir ngan can kha nang cgng sinh ciia na A M vao cay chu. So lieu thu dugc tiong nghien ciiu nay cd thi dugc sir dung nhu budc khdi dau cho nhung nghien ciiu tiep sau do tren nhiing quy md khac nhau va vdi nhiing dli tugng thuc vat khac nhau.
6 5 - 5?
Hieu suat
0
B Hieu suat XLP if cua cac cay thf ngtiiem
-
H i -
TN1 TN2 TN4 TIMS TN6 TN7 TN8
Hinh 6. Hieu suat xir li chi cua cac cay thi nghiem
4. KET LUAN
Dat ngheo dinh duong (P: 252,3 ppm, N: 44.1 ppm) kich thich su cpng sinh cua nim AM vao cay ngo (TNI: 22,8%), ngugc lai dat giau dinh duong (P: 952,3 ppm, N: 105 ppm) lai kim ham su cgng sinh nay (TN8: 5,13%)). Sir cgng sinh cua nam AM vdi mat dp ldn giiip cho cay sinh trudng va phat trien tot va hap thu lugng ldn kim loai vao sinh khdi cay dac biet d bp re (lugng Pb tich tu tiong re TNI gap 1,5 lan so vdi TN8) va ham lugng Pb tich liiy d ri cao hon 6 den 8 lan so vdi than la d tat ca cac cong thiic thi nghiem. C5 ham lugng P la 752,5 ppm va N:
87,6 ppm trong dat cho cay dat hieu suat xii li chi trong dit cao nhit.
Ttr cac kit qua nghien ciiu tren cho thay mudn thu nhan sinh khii nim AM trong dit thi nen trdng ngd tien dit ngheo dinh duong nito va photpho, nhung dl xii ly dit d nhiim chi thi khdng nen bdn qua nhieu phan nito va photpho.
TAI LIEU THAM KHAO
1. Vien thd nhudng ndng hoa - So tay phan tich dit, nudc, phan bdn cay tring I, Nha xuit ban Ndng nghiep, 1998, tr. 100-105.
2. Andre Fortin, Guillaume Becard, Stephane Deleck, Yolande Dalpe, Marcst-Amamd, Andrew P Coughlan, and Yves Piche - Mycorrhiza on root-organ cultures, NRC Reseach Press. Can. L Bot. 80 (2002) 2-15
3. Leyval, C, Singh, B. R., Janer E. J. - Occurrence and infectivity of arbuscular mycorrhizal fungi in some Norwegian soils influenced by heavy metals and soil properties, Water, Air Soil Pollut. 83 (1995) 203-216.
4. Mahendra Rai, Ajit Varma - Arbuscular Mycorrhiza-like biotechnological potential of Piriformospora indica which promotes the growth of Adhatoda vasica Nees, Plant Biotechnology 8 (1) (2005) 23-28.
5. Neil Willey - Phytoremediation: Methods and Reviews, Humana Press Inc. Vol. 23, 2007, 15-17.
6. Pennanen T., Frosetgerd A., Fritze H., Barth E. - Photpholipid fatty acid composition and heavy metal tolerance of soil microbial communities along two heavy metal polluted gradients in coniferous forests, Appl Environ Microbiol, 62 (1996) 420-428.
7. Pawlowska T. E., Blaszkowski J., Ruhling A. - The mycorrhizal status of plants colonizing a calamine spoil mound in southern Poland, Mycorrhiza 6 (1996) 499-505.
8. Shetty K. G., Banks M. K., Hetrick B. A., Schwab A. P. - Effects of mycorrhizae and fertihzer amendments on zinc tolerance of plants. Environ. Pollut. 88 (1995) 307-314.
SUMMARY
THE INFLUENCE OF PHOSPHOROUS AND NITROGEN CONTENT IN SOIL ON THE SYMBIOSIS ABILITY OF ARBUSCULAR MYCORRHIZAS FUNGI IN THE HOST PLANT
ROOT, AND THE EFFECT OF LEAD REMOVAL FROM SOIL BY MAIZE
The study was conducted to investigate how the nutiient factor in soil (Phosphorous and Nitrogen) influence on the symbiosis ability of arbuscular mycorrhizas flingi to the host plant, and the effect of lead (Pb) removal from soil by maize. The results showed that, AM fungi were inhibited to penetrate into maize root when they were cultured in rich nutrient soil. In the soil with low concentration of nitrogen and phosphorus nutrient (formula TNI), the density of AM fiingi was three times higher than this in high nutrient soil (formula TN8), and the amount of Pb accumulate in maize biomass of TNI was also the highest in all formulas. However, the treatment effect of TNI formula did not get the optimal value, because the total amount of biomass was the lowest in comparison with the other formulas. Formula TN5 gained the optimal value of treatment process, since they could remove the highest amount of lead out from the soil.
The concentration of phosphorus and nitrogen in formula TN5 were 752.5 ppm and 87.6 ppm, so they did not prevent AM fungi from inoculating maize root, and they were not too low for maize to achieve high biomass.
Bia chi: Nhgn bdi ngdy 8 thdng 8 ndm 2009 Vien Cdng nghe Mdi trudng,
Vien Khoa hgc va Cdng nghe Viet Nam.
