Tap chi Khoa hoc va Cong nghe Nong nghiep Viet Nam - So 05(126)/2021 PHAN LAP, TUYEN CHON VA DINH DANH VIKHUAN HOA TAN LAN, CO DINH DAM VA TONG HOP IAA VUNG RE CAY DINH LANG (P o iyscia s fr u tic o s a ) Nguyen Quoc Khuong1, Tran Ngoc Hflu1, Le Vinh Thuc1, Le Thi My Thu1, Nguyen Hong Hue1, Tran Chi Nhan2, Pham Duy Tien2, Ly Ngoc Thanh Xuan2 TOM TAT Muc tieu cua nghien cdu la xac dinh diipc vi khuan co kha nang hoa tan lan, co dinh dam va tong hpp IAA vung re cay dinh lang Miidi ba mau dat vung re dinh lang thu thap tai huyen Tri Ton, tinh An Giang diipc sd dung de phan lap vi khuan hoa tan lan tren moi trUdng NBRIP Ket qua nghien cdu xac dinh diipc 30 dong vi khuan hoa tan lan, 15 dong co kha nang chiu dvfng diipc m i tnidng chua va dong vi khuan ky hieu AC10L2 co hoat tinh hoa tan lan cao nhat, dat ham liipng lan tan 20,5 mg P L Dong vi khuan AC10L2 cung co kha nang co dinh dam va tong hop IAA, vdi ham liipng lan loot la 63,2 mg P L va 0,81 mg IAA L I Dong vi khuan AC10L2 dilpc dinh danh la Bacillus subtilis bang ky thuat 16S rDNA Td khoa: Dinh lang (Poiyscias fruticosa L Harms), vi khuan co dinh dam, vi khuan hoa tan lan, vi khuan tong hop IAA, Bacillus subtilis I DAT VAN DE Dinh lang la mot loai thuoc ho Araliaceae, co nguon goc tit An Do Cay phat trien tot nhat d nhiet tit 19 - 29°C va nhay cam vdi nhiet cao (Pandya et aL, 2020) Theo Nguyen va cong tac vien (2020), dinh lang la cay thao ditctc co tac dung y hoc, dac biet la chdng oxy hoa, chong viem va sot Lan (P) dong mot vai tro quan trong qua trinh sinh tnldng va phat trien cua cay Tuy nhien, nhieu loai dat diiOc xac dinh ngheo P hiiu dung cho cay vi bi co dinh bdi sat, nhom va canxi de tao cac hop chat khong tan cay khong the hap thu ditOc (Weil and Brady, 2017), P tong so co the van cao (Nussaume et al, 2011) Lan tham gia vao mot so chdc nang cay nhtl quang hop, ho hap, tong hop acid nucleic, tao nang ldOng, mot phan khong the thieu cua phosphoprotein va phospholipid (Cordell et al., 2011) Dam (N) co the bo sung tii nhieu nguon khac nhii khoang hoa, c6 dinh N2 va phan bon (Zhang et al., 2017) Tuy nhien, sau thcfi gian dai bon phan hoa hoc, tinh trang thoai hoa va nhiem dat cang tram (Gurdeep and Reddy, 2015), dong thdi gia tang phat thai khl nha klnh gay nhiem moi triibng (Nguyen Quoc KhiiOng, Ngo Ngoc Hhng, 2014) Indole-3acetic acid (IAA), dong vai tro quan trong viec dieu hoa sinh triidng cay (Jin et al., 2021) Wei va cong tac vien (2018) cho biet, cac vi sinh vat dat gop phan gia tang lupng lan hdu dung dat cho cay Ngoai ra, vi khuan cung dong vai tro cung cap dam cho cay (Nguyen Quoc KhifOng 1Bo mon va ctv., 2019) Nghien cdu cua Singh va cong tac vien (2016) cung cho biet, vi khuan vung re da dtipc sd dung de tang ciidng sinh triidng, phat trien cay Muc tieu cua nghien ciiu la tuyen chon vi khuan vung re cay dinh lang co kha nang hoa tan lan, co dinh dam va tong hpp IAA II vAt l ie u vA p h UOn g p h Ap n g h ie n c tfu 2.1 Vat lieu nghien cdu Mau dat cho phan lap vi khuan diipc thu tai ba xa thuoc huyen Tri Ton, tinh An Giang Moi triidng NBRIP (g/L) gom: 10 g glucose, g Ca3(P 4), 2,5 g MgCl2-6H20 , 0,25 g MgS0 4-7H20 , 0,2 g KC1,0,1 g (NH 4)2S 4, diipc sd dung de phan lap vi khuan vung re Moi triidng Burk’s (g/L) gom: 10 sucrose, 0,41 KH 2P 4, 0,52 KH2P 4, 0,05 Na2S04, 0,2 CaCl2, 0,1 M g S - H 20 , 0,005 F e S *7H 20 , 0,0025 Na2M o0 4‘2H20 va 20 agar, diipc sd dung de nuoi cac dong vi khuan va danh gia kha nang co dinh dam 2.2 PhiiPng phap nghien cdu 2.2.1 Thu thap va phan tich m&u dat MiiPi ba mau dat vdng re ddpc thu tai cac vi tri xung quanh goc cua cay dinh lang nam tuoi, vdi khoi ldpng 500 g/mau, diipc td lieu hoa, trd lanh va phan tich cac chi tieu pH H2o, pH Kci> lan tong so, lan de tieu theo phiiPng phap diipc mo ta bdi Sparks va cong tac vien (1996) tai phong thi nghiem cua Khoa hoc cay trong, Khoa Nong nghiep, Triidng Dai hoc Can Thd -TrUdng Dai hoc An Giang; Tnidng Dai hoc Quoc gia Ho Chi Minh Tap chi Khoa hoc va Cong nghe Nong nghiep Viet Nam - So 05(126)/2021 Khoa Nong nghiep, trbbng Dai hoc Can Thd va Khu thi nghiem-thbc hanh, trbbng Dai hoc An Giang 2.2.2 Phan lap, tuyen chon vi khuan hoa tan lan, co dinh dam va tong hdp IAA Vi sinh vat hoa tan lan dbdc phan lap theo phitdng phap dbdc mo ta bbi Nguyen Quoc Khbdng va cong tac vien (2019) va dinh liidng hoat tinh hoa tan lan theo phitdng phap mo ta bbi Cao Ngoc Diep va Nguyen Thi Mong Huyen (2015) Kha nang chiu dbdc moi trbbng chua cua cac vi khuan hoa tan lan phan lap dbdc danh gia bang each nuoi cay moi trbbng co pH = 5,5 dat gia tri OD660 = 0,5 Lay 0,5 mL sinh khoi vi sinh vat cho vao ong nghiem chba san 4,5 mL moi tritctng NBRIP long, u 48 gict dieu kien toi, sau lay mL dung dich tren may so mau bdbc song 660 nm Cac dong vi khuan dat gia tri OD660 lbn hdn 0,7 la cac vi sinh vat co kha nang chiu dildc moi tritctng chua (Nguyen Quoc Khitdng va ctv., 2019) Kha nang co dinh dam, tong hdp IAA cua cac dong vi khuan hoa tan lan ditdc thbc hien theo phitdng phap ditdc mo ta beti Cao Ngoc Diep va Nguyen Thi Mong Huyen (2015) Tat ca cac thi nghiem danh gia kha nang chiu moi trbbng chua, hoa tan lan, co dinh dam va tong hdp IAA dildc thbc hien dieu kien toi, vefi lan lap lai 2.2.3 Dinh danh vi khudn Cac dong vi khuan co hoat tinh hoa tan lan, co dinh dam, tong hdp IAA cao dildc nuoi moi tritdng NBRIP 48 gib, ly tam b toe 10.000 vong/phut phut nham thu dildc te bad de ly trich DNA bang Genomic DNA Prep Kit (BioFACT™), theo hilbng dan cua nha san xuat Sau kiem tra nong va thuan tren 1,0% w/v agarose gel bang dien di San pham DNA ditdc khuech dai gen ma hoa 16S rRNA bang ky thuat PCR vbi cap moi F va 1492R (Turner et al, 1999) nhil mo ta iP ro o f High-Fidelity PCR Kit - Bio-Rad (BioRad, Hercules, CA) bbi T100TM thermo cycler (BioRad) cho vi khuan vung re So kich thilbe cua san pham PCR vbi thang DNA chuan de xac nhan vi tri cac band kich thildc 1.500 bp doi vbi vi khuan vung re San pham PCR ditdc tinh sach bang TLANquick Midi Purification Kit (Tiangen Biotech Ltd., Beijing, China) theo hilbng dan nha san xuat Sau do, kiem tra lai thuan tren 1,0% w/v agarose gel bang dien di San pham PCR da tinh sach dildc giai trinh tit bang may giai trinh tit tit dong tai Macrogen DNA Sequencing Service (Macrogen, Seoul, Korea) Ket qua giai trinh tit vbi sac ditdc phan tich bang phan men BioEdit, phien ban 7.0.5.3 (Hall, 1999) va phan men ChromasPro version 1.7 (http://technelysium.com.au/wp/chromaspro) Giai trinh tit cua cac dong vi khuan ditdc so sanh vbi cac trinh tit co san ngan hang gen bang cong cu Basic Local Alignment Search Tool (BLAST) cua National Center for Biotechnology Information (NCBI) de xac dinh mite titdng dong So lieu nghien ebu dildc xb ly thong ke bang phan mem SPSS phien ban 16.0 2.3 Thbi gian va dia diem nghien ebu Thi nghiem dbdc thiic hien tb thang 08 nam 2019 den thang nam 2020 tai Khoa Nong nghiep, trbbng Dad hoc Can Thd va Khu thi nghiem-thiic hanh, tnlbng Dai hoc An Giang III KET QUA VA THAO LUAN 3.1 Dac tin h dat phan lap vi khuan hoa tan lan vung re cay dinh lang Dat dinh lang tai xa Chau Lang, An Cit va Liidng Phi co chua tiem tang b mbc trung tinh vbi pH KC1 dao dong 6,46 - 7,68 Ham lbdng lan tong so dat khoang 0,05 - 0,10% cho thay ham lbdng lan tong so dat cua xa b mbc trung binh (Nguyen Xuan Cit, 2000) Ham lbdng lan de tieu dat 22,8 - 90,2 mg P k g va dbdc danh gia b mbc trung binh den cao (Horneck et al., 2011) (Bang 1) Do do, dat khong the cung cap du dbbng chat lan cho cay Chinh vi vay, vi khuan hoa tan lan co the giup cay dat dbdc sinh khoi toi da Bang Dac tinh dat phan lap vi khuan vung re hoa tan lan dinh lang thu tai huyen Tri Ton, tinh An Giang 7,68 ± 0,53 P tong so (%) 0,10 ± 0,02 P de tien (mgPkgr') 90,2 + 813 6,83 + 1,08 0,05 ± 0,06 2Z»±2XJ | 6,46 ± 0,61 0,05 ± 0,04 593 ± W fi X So mau P^KCl (D at: KC1 ~ 1:5) Chau Lang An Cb Lbdng Phi Dia diem Tap chiKhoa hoc va Cong nghe Nong nghiep Viet Nam - 56 05(126)/2021 3.2 Tuyen chon vi khuan hoa tan lan khuan LP03L3 co kha nang hoa tan lan thap nhat chi dat 0,83 mg/L Cac dong vi khuan lai tao ham liidng lan tan khoang 4,13 -17,8 mg/L Tti 13 mau dat vung re cay dinh lang 02 nam tuoi, tong so 30 chung vi khuan co kha nang hoa tan lan diidc phan lap va lam thuan Ket qua nuoi cay cac dong vi khuan phan lap moi triicmg NBRIP long co pH =5 ghi nhan dong vi khuan co gia tri OD660 nho hdn 0,5; 17 dong vi khuan co gia tri OD660 khoang 0,5 - 1,0 va dong co gia tri OD660 > 1,0.15 dong vi khuan co gia tri OD660 > 0,7 dtidc danh gia co kha nang thich dng m i triicing chua va diiqc sd dung de danh gia dinh liidng kha nang hoa tan lan (Hinh 1) Ket qua danh gia kha nang hoa tan lan cua 15 dong vi khuan vung re chiu diicfc moi tnldng the hien hinh cho thay mdc hoa tan lan gifla cac dong vi khuan khac biet co y nghia thong ke 5%, do, dong vi khuan ky hieu AC10L2 co kha nang hoa tan lan cao nhat, dat 20,5 mg/L Dong vi 1,00 Hinh Kha nang song cua cac chung vi khuan da lam thuan dieu kien pH chua 24,0 91 18,0 be be 12.0 ^5 6.0 X f _fiL 0.0 ^ ^