Nghiên cứu vi khuẩn nội sinh lúa nhằm ứng dụng trong phòng trị bệnh thối rễ do dickeya zeae gây ra

ĐẠI HỌC QUỐC GIA HÀ NỘI TRƯỜNG ĐẠI HỌC KHOA HỌC TỰ NHIÊN - NGUYỄN DUY TỚI NGHIÊN CỨU VI KHUẨN NỘI SINH LÚA NHẰM ỨNG DỤNG TRONG PHÒNG TRỊ BỆNH THỐI RỄ DO Dickeya zeae GÂY RA LUẬN VĂN THẠC SĨ KHOA HỌC Hà Nội ‒ 2021 ĐẠI HỌC QUỐC GIA HÀ NỘI TRƯỜNG ĐẠI HỌC KHOA HỌC TỰ NHIÊN - NGUYỄN DUY TỚI NGHIÊN CỨU VI KHUẨN NỘI SINH LÚA NHẰM ỨNG DỤNG TRONG PHÒNG TRỊ BỆNH THỐI RỄ DO Dickeya zeae GÂY RA Chuyên ngành: Vi sinh vật học Mã số: 8420101.07 LUẬN VĂN THẠC SĨ KHOA HỌC NGƯỜI HƯỚNG DẪN KHOA HỌC: TS ĐINH THÚY HẰNG PGS TS PHẠM THẾ HẢI Hà Nội – 2021 Nguyễn Duy Tới – Cao học K27 LỜI CẢM ƠN Lời đầu tiên, tơi xin bày tỏ lịng kính trọng gửi lời cảm ơn chân thành tới TS Đinh Thúy Hằng, PGS TS Phạm Thế Hải, TS Nguyễn Kim Nữ Thảo người tận tình hướng dẫn tơi q trình thực đề tài, giúp tơi hồn thành tốt luận văn Tôi mong muốn gửi lời cảm ơn chân thành tới Ban lãnh đạo cán Viện Vi sinh vật Công nghệ Sinh học, Đại học Quốc Gia Hà Nội tạo điều kiện thuận lợi trang thiết bị sở vật chất cho tơi hồn thành nội dung nghiên cứu Tôi xin cảm ơn đề tài NĐT.34.ITA/17: “Nghiên cứu hệ vi sinh vật nội sinh phục vụ sản xuất chế phẩm phòng chống bệnh bạc (Xanthomonas oryzae pv oryzae) bệnh thối rễ (Dickeya zeae) lúa” tạo điều kiện cho tham gia thực nội dung nghiên cứu Tôi xin bày tỏ lòng biết ơn chân thành tới thầy cô giáo, cán khoa sinh học, Trường Đại học Khoa học Tự nhiên, Đại học Quốc gia Hà Nội giúp đỡ trang bị kiến thức hữu ích cho tơi thời gian học tập trường Cuối cùng, tơi xin bày tỏ lịng biết ơn sâu sắc tới gia đình, bạn bè, anh chị đông nghiệp cổ vũ, động viên vượt qua khó khăn q trình học tập nghiên cứu Hà Nội, ngày……tháng……năm 2021 Học viên Nguyễn Duy Tới Chuyên ngành Vi sinh vật học MỤC LỤC MỞ ĐẦU Mục tiêu đề tài CHƯƠNG 1: TỔNG QUAN TÀI LIỆU 1.1 Vi khuẩn Dickeya zeae bệnh thối thân rễ trồng 1.1.1 Lúa vai trò lúa đời sống 1.1.2 Đặc điểm phân loại vi khuẩn Dickeya zeae 1.1.2 Phân bố tự nhiên phương thức gây bệnh 1.1.3 Bệnh thối thân/gốc Dz biện pháp phòng trị 1.2 Vi khuẩn nội sinh thực vật (EB) lợi ích chủ 10 1.2.1 Đa dạng EB chủ 10 1.2.2 Cơ chế xâm nhập phân tán 11 1.2.3 Vai trò EB chủ 13 CHƯƠNG 2: VẬT LIỆU VÀ PHƯƠNG PHÁP 20 2.1 Nguyên vật liệu 20 2.1.1 Mẫu lúa chủng vi khuẩn kiểm định 20 2.1.2 Hóa chất 20 2.1.3 Thiết bị, dụng cụ 20 2.2 Phương pháp nghiên cứu 21 2.2.1 Phân lập vi khuẩn nội sinh 21 2.2.2 Đánh giá hoạt tính kháng Dickeya zeae (Dz) 22 2.2.3 Tách DNA genome từ chủng vi khuẩn khiết 22 2.2.4 Giải trình tự gen 16S rDNA dựng phân loại 23 2.2.5 Thí nghiệm in planta đánh giá khả đối kháng Dz vi khuẩn nội sinh 24 2.2.6 Tách chiết xác định hoạt chất sinh học kháng Dz 26 2.2.7 Nghiên cứu đặc điểm hình thái sinh lý vi khuẩn nội sinh 27 2.3 SƠ ĐỒ THÍ NGHIỆM 28 CHƯƠNG 3: KẾT QUẢ VÀ THẢO LUẬN 29 3.1 Phân lập vi khuẩn nội sinh từ lúa 29 3.2 Sàng lọc vi khuẩn nội sinh có hoạt tính kháng Dickeya zeae (Dz) 30 3.3 Xác định vị trí phân loại chủng có hoạt tính kháng Dz cao 32 3.3.1 So sánh trình tự gen 16S rDNA 32 3.3.2 Nghiên cứu đặc điểm hình thái 34 3.4 Xác định hoạt chất kháng Dz chủng EB có tiềm cao 35 3.4.1 Tinh hoạt chất kháng Dz HPLC 35 3.4.2 Nghiên cứu xác định cấu trúc hoạt chất kháng Dz từ chủng VY81 36 3.5 Đánh giá hiệu chủng VY03 đối kháng Dz lúa 39 3.5.1 Xác định điều kiện ảnh hưởng đến sinh trưởng chủng VY03 39 3.5.2 Thí nghiệm in planta đánh giá hiệu ức chế Dz chủng VY03 40 KẾT LUẬN 43 KIẾN NGHỊ 43 TÀI LIỆU THAM KHẢO 44 PHỤ LỤC 57 TÓM TẮT 74 DANH MỤC CÁC HÌNH Hình 1.1 Hình thái tế bào vi khuẩn D zeae kính hiển vi điện tử quét [76] .5 Hình 1.2 Bệnh thối gốc Dz lúa (A), ngô (B) chuối (C) [25, 80, 103] .8 Hình 1.3 Các đường xâm nhập EB vào táo [42] 12 Hình 1.4 Điều hịa phytohormone thực vật nhờ EB [128] 16 Hình 3.1 Phân lập vi khuẩn nội sinh lúa (A) đĩa cấy gạt từ mẫu rễ sau khử trùng bề mặt; (B) Đĩa kiểm chứng đặt trực tiếp mẫu thân, rễ, sau khử trùng bề mặt; (C) Đĩa cấy gạt nước rửa mẫu rễ lần cuối sau khử trùng bề mặt 29 Hình 3.2 Tế bào chủng DZ2Q qua kính hiển vi phản pha (A) lúa Bắc Thơm bị lây bệnh nhân tạo từ chủng DZ2Q viện Bảo vệ Thực vật (B) 30 Hình 3.3 Hoạt tính kháng Dz chủng VY03, VY81, VY149, VY166, VY235, VY244 đánh giá phương pháp khuyếch tán đĩa thạch 32 Hình 3.4 Cây phát sinh chủng loại neibourgh joining dựa trình tự 16S rDNA gần đủ chủng nội sinh VY03 (A); VY81 (B); VY65, VY149, VY166, VY235, VY244 (C) so sánh với lồi có quan hệ gần gũi Trong cây, chủng L acidophilus, B gladioli, B cepacia tương ứng chọn làm out group 33 Hình 3.5 Hình thái tế bào khuẩn lạc chủng VY03 (A, B), VY81 (C, D) 34 Hình 3.6 Phổ hấp phụ bước sóng 240 nm hoạt chất thô tách từ chủng VY03 (A); VY81 (B) 35 Hình 3.7 Tách chiết hoạt chất từ canh trường chủng VY81 (A) Hiệu suất tách chiết dung mơi khác (B) Hoạt tính kháng Dz thời điểm ni cấy khác 36 Hình 3.8 Phổ hấp thụ UV - vis hoạt chất kháng Dz tinh từ chủng VY81 (A) so sánh với phổ hấp thụ chất 2-(2-heptenyl)-3-methyl-4-quinolinol (B) [58] 37 Hình 3.9 Cấu trúc hóa học (A) 2-(2-heptenyl)-3-methyl-4-quinolinol [58] (B) 2-(2heptenyl)-3-methyl-4(1H)-quinolone [123] .38 Hình 3.10 Ảnh hưởng điều kiện nuôi cấy tới sinh trưởng chủng VY03 40 Hình 3.11 Thí nghiệm in planta đánh giá hiệu ức chế Dz gây bệnh thối gốc/thân lúa chủng VY03 (A) Công thức 1; (B) Công thức 2; (C) Công thức 3; (D) Cơng thức 41 Hình 12 Vết thương tổn nhiễm Dz (A); điều trị chủng VY03 (B) độ dài vết thương bị bệnh (C) 42 DANH MỤC CÁC BẢNG Bảng 1.1 Các loài Dickeya chủ chúng Bảng 1.2 Một số kháng sinh sản xuất EB ức chế mầm bệnh thực vật 17 Bảng 2.1 Phản ứng PCR khuếch đại gen 16S rDNA 24 Bảng 2.2 Thành phần dung dịch Hoagland (dung dịch mẹ 2×)[142] .25 Bảng 2.3 Thành phần dung dịch vi lượng (1 lít) 25 Bảng 2.4 Thành phần môi trường Landy [159] 27 Bảng 3.1 Số lượng vi khuẩn nội sinh phân lập từ mẫu lúa 30 Bảng 3.2 Kết sàng lọc tìm kiếm chủng vi khuẩn nội sinh lúa kháng Dz 31 DANH MỤC CÁC KÝ HIỆU VÀ CHỮ VIẾT TẮT ACC Aminocyclopropane-1-carboxylate ACN Acetonitril Bp Base pair BSA Bovin serum albumin CI Chloroform-isoamyl alcohol CFU Colony Forming Unit (Đơn vị khuẩn lạc) DGGE Denaturing Gradient Gel Electrophoresis DNA Deoxyribonucleic acid dNTP Deoxyribonucleotide triphosphate Dz Dickeya zeae EB Endophytic Bacteria EDTA Ethylenediaminetetraacetic acid FISH Fluorescence in situ hybridization IAA Indole-3-acetic acid ISR Induced Systemic Resistance MQ Mili-Q NMR Nuclear Magnetic Resonance OD Optical Density PCR Polymerase Chain Reaction rDNA Ribosomal Deoxyribonucleic Acid SDS Sodium Dodecyl Sulfate SEM Scanning Electron Microscope TAE Tris-Acetic-EDTA (đệm) Taq Thermus aquaticus polymerase TE Tris-EDTA (Đệm) TGGE Temperature Gradient Gel Electrophoresis TKT Thực khuẩn thể T-RFLP Terminal restriction fragment length polymorphism TTSS Type three secretion system VOC Volatile organic compound (Hợp chất hữu dễ bay hơi) VSV Vi sinh vật Nguyễn Duy Tới – Cao học K27 MỞ ĐẦU Theo thống kê, sản lượng gạo toàn giới năm 2018 đạt 769.9 triệu tấn, nguồn lương thực giới [35] Thiệt hại hàng năm dịch bệnh gây lúa nước châu Á ước tính từ 24-41%, đó, bệnh bạc lá, đạo ơn, đốm nâu gây tổn thất nghiêm trọng [129] Bệnh thối gốc/thân vi khuẩn Dickey zeae (tên trước Erwinia chrysanthemi pv zeae) gây báo cáo Prasad (1930) với biểu xuất vết thối màu nâu sậm gần gốc, bẹ ngả vàng, cháy khơ, đổ gục [76] Sau đó, Dickeya zeae ghi nhận nguyên nhân gây thối rễ lúa Nhật Bản, Trung quốc, Đài Loan [51, 82, 117] Ở Việt Nam, bệnh thối gốc lúa Dickeya zeae gây ghi nhận Tiểu Cần, Trà Vinh sau lan rộng nhiều tỉnh khác đồng sông Cửu Long [1] Sử dụng thuốc bảo vệ thực vật để kiểm sốt bệnh vi sinh vật (VSV)/cơn trùng gây biện pháp áp dụng nhiều khu vực canh tác nông nghiệp giới Lạm dụng thuốc bảo vệ thực vật dẫn đến ảnh hưởng xấu cho môi trường, chưa kể tới việc xuất VSV kháng thuốc làm giảm hiệu phòng chống bệnh [164] Kiểm soát sinh học nhờ sử dụng vi sinh vật đối kháng với mầm bệnh hướng nhiều quốc gia quan tâm phát triển nông nghiệp bền vững, giúp thay phần thuốc bảo vệ thực vật [49] Nhiều loài vi khuẩn Bacillus, Burkholderia, Lysobacter, Pantoea, Pseudomonas Streptomyces nghiên cứu sử dụng để kiểm soát tác nhân gây bệnh trồng [46] Nhiều loài thuộc chi nêu vi khuẩn nội sinh thực vật, ngồi việc đóng vai trị tác nhân kiểm sốt sinh học, chúng cịn đem lại nhiều lợi ích cho trồng tăng cường thu nạp dinh dưỡng thúc đẩy tăng trưởng [9] Xuất phát từ nhu cầu thực tiễn, thực đề tài: “Nghiên cứu vi khuẩn nội sinh lúa nhằm ứng dụng phòng trị bệnh thối rễ Dickeya zeae gây ra” để tìm lồi vi khuẩn nội sinh hoạt chất sinh học chúng đối kháng vi khuẩn gây bệnh Dickeya zeae nhằm ứng dụng nông nghiệp hữu 10 Chuyên ngành Vi sinh vật học GAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTAGTTTG TTGGGGATTCATTTCCTTAGTAACGTAGCTAACGCGTGAAGTTGACCGCCTGGGGGAG TACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAT GATGTGGATTAATTCGATGCAACGCGAAAAACCTTACCTACCCTTGACATGGTCGGAA CCTTGGAGAGATCCGAGGGTGCTCGAAAGAGAACCGATACACAGGTGCTGCATGGCT GTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGT CCTTAGTTGCTACGCAAGAGCACTCTAGGGAGACTGCCGGTGACAAACCGGAGGAAG GTGGGGATGACGTCAAGTCCTCATGGCCCTTATGGGTAGGGCTTCACACGTCATACAA TGGTCGGAACAGAGGGTCGCCAACCCGCGAGGGGGAGCTAATCCCAGAAAACCGATC GTAGTCCGGAT Phụ lục 3: Các cơng trình khoa học tác giả liên quan đến luận văn ISOLATION OF RICE ENDOPHYTIC BACTERIAL STRAIN VY81 AND STUDY ITS BIOACTIVE COMPOUND ANTAGONIZING THE PHYTOPATHOGEN DICKEYA ZEAE Nguyen Duy Toi1, Dinh Thi Ngoc Mai2, Nguyen Thi Hieu Thu1, Nguyen Tien Dat3, Nguyen Kim Nu Thao4, Dinh Thuy Hang1* VNU Institute of Microbiology and Biotechnology, Vietnam National University Hanoi Faculty of Biotechnology, Chemistry and Environmental Engineering, Phenikaa University Center for Research and Technology Transfer, Vietnam Academy of Science and Technology Faculty of Biology, Hanoi University of Science, Vietnam National University Hanoi SUMMARY The bacterial communities performing endophytic lifestyle have been proven to possess a number of characteristics useful to host plants and thus are considered as “plant probiotics” Many probiotic bacteria were reported for antagonism against different plant pathogens, including bacteria, fungi, and nematodes The use of endophytic bacteria as biocontrol agents would have great potentials, allowing reducing the use of agrochemicals and thus support a sustainable agriculture In this study, endophytic bacteria isolated from rice plants of IR4625 cultivar from Long An province, Vietnam were used for screening strains that have antagonistic activity against Dickeya zeae (Dz), the bacterium causing foot rot disease The rice plants had foot rot disease symptoms, i.e dark-brown foot with odor smell typical for bacterial infection Strain VY81 was isolated from a surface sterilized rice stem sample adjacent to the foot rot area The crude extract of strain VY81 showed significant antagonistic activity against Dz, with the inhibition zone of 14,25 mm ± 1,06 in diameter Strain VY81 produced the compound antagonizing Dz at maximal level after 48 h cultivated in TSB medium The activity was found mainly in the culture broth, just a small part was found intracellularly The bioactive compound antagonizing D zeae produced by strain VY81 was purified by HPLC and analyzed by mass spectrometry and NMR spectroscopy The compound was identified as a quinoline alkaloid, the chemical formula is C 17H21ON with chemical name 2-(2-heptenyl)-3-methyl-4(1H)-quinolone Comparative analyses of the 16S rDNA gene sequence revealed that strain VY81 belonged to the genus Burkholderia, most closely related to Burkholderia cepacia (99,77% sequence homology) The 16S rDNA sequence of strain VY81 was deposited at GenBank under accession number MW056196 Strain VY81 and its quinolone compound would have application potential for development of biocontrol product against the foot rot disease caused by Dickeya zeae Keywords: Biocontrol, Burkholderia cepacia, Dickeya zeae, endophytic bacteria, quinoline, rice foot rot disease INTRODUCTION The genus Dickeya includes pectin-utilizing species, belonging to the family Enterobacteriaceae Dickeya spp cause disease in monocot and dicot plants (Samson et al., 2005; Ma et al., 2007) Dickeya zeae (Dz, former Erwinia chrysanthemi pv zeae) has been identified as the cause of rice foot rot, corn stalk rot, banana and ornamental plant soft rot in various regions of the world (Pu et al., 2012; Bertani et al., 2013; Zhang et al., 2014; Kumar et al., 2017; Hu et al., 2018) Corn stalk rot has been reported in the US, Brazil, France, Italy, Senegal, Cuba, Egypt, Mexico, India, Korea, Iran, Japan, China and Thailand (Li et al., 2020) Rice foot rot disease has mainly occurred in southern China resulting in ∼ 10 to 30%, even 60% losses ofrice yield The disease threatens other rice growing regions in Southeast Asian and European countries (Bertani et al., 2013; Hu et al., 2018) Banana soft rot disease caused by Dz has been found in Ivory Coast, Jamaica, Panama and Martiniue (Samson et al., 2005; Hu et al., 2018) In China, soft rot disease has become serious in over 6000 of banana plantation from 2009 to 2012 (Lin et al., 2010; Zhang et al., 2014) The natural host range of Dz has been extended to hyacinth and lily (Jafra et al., 2009; Hu et al., 2018) The main virulence factors of Dickeya spp are cell wall degrading enzymes that lead to soft rot This phytopathogen can be spread out through water, stays on weeds and plant residues, leading to difficulty in controlling the disease (Samson et al., 2005) In Vietnam, Dz mainly attacks rice and dragon fruit trees with symptoms such as stem rot, green wilt (Nguyen Van Hoa et al., 2015; Tran Hung Minh et al., 2016) Rice foot rot was first reported in Tieu Can, Tra Vinh in 2000, and then it spread rapidly throughout the Mekong Delta provinces The infected rice plants had rotten roots and stalks that turned dark brown with foul odor, leading to the loss of a clump or the whole field (Tran Hung Minh et al., 2016) Application of biological control agents in the fight against microbial phytopathogens (bacteria and fungi) has become attractive in terms of reducing the use of agrochemicals (O'Brien 2017) Jafra et al (2009) reported that Rahnella aquatilis and Erwinia persicinus had high antagonistic activity against Dz infected hyacinth Recently, Li et al (2020) demonstrated that B subtilis A2 isolated from root of the guzmania rondo tree had a suppressing effect on stinging rot of host plant Other bacterial strains, i.e Pseudomonas fluorescens SC3, P parafulva SC11 and Bacillus velezensis 3–10 isolated from healthy root and stem of cabbage, ginger, banana, rice, as well as rhizosphere soil were also shown to be new potential biological control agents against Dz (Li et al., 2020) In Vietnam, only a few researches on biological antagonistic activity of microbial strains against Dz causing rice foot rot have been published Tran Vu Phuong and Phung Thi Thanh Thao (2015) reported three strains Bacillus sp B57, B54 and B128.2 that were able to inhibit Erwinia chrysanthemi with the inhibitory zone of 8.14 mm, 7.57 mm and 7.52 mm in radius, respectively In another study, Tran Hung Minh et al (2016) investigated the effects of bacteriophages on 14 strains of E chrysanthemi isolated from rice rot roots The study has identified 35 bacteriophages parasitizing 14 strains of E chrysanthemi, among those bacteriophages showed multi-host infection Of special interest was the bacteriophage ΦEchKG8b that showed the most efficiency in controlling the disease under net house conditions (Tran Hung Minh et al., 2016) In this study, we isolated rice endophytic bacteria and selected strains that have antagonistic activity against Dz for application in controlling the foot rot disease The endophytic strain VY81 isolated from stem of a rice plant with foot rot disease was studied in details since it had prominent antagonistic activity against Dz The taxonomic position of the strain was identified based on 16S rDNA sequence comparative analyses and the Dz inhibiting bioactive compound produced by the strain was purified and its chemical structure was determined MATERIAL AND METHODS Materials Rice plants (cultivar IR4625) were harvested in Spring-Summer season 2018 in Long An province for the isolation of endophytic bacteria The strain Dickeya zeae DZ2Q was kindly provided by Professor Vittorio Venturi (International Center for Genetic Engineering and Biotechnology, Trieste, Italy) Isolation of rice endophytic bacteria Rice plants were washed carefully under tap water and the upper stems were cut into segments of 500 - 1000 mg The stem samples were surface sterilized by submerging successively in 75% ethanol for min, then in 50% hypochlorite (7% of active Cl) for and again in ethanol 75% for Between each of these surface sterilization steps and at the end of the treatment procedure, the samples were rinsed carefully with sterile water (5 times) Efficiency of the surface sterilization procedure was controlled by plating 0.1 ml of the final wash as well as a piece of the sterilized roots on Tryptic Soy Agar (TSA) plates and checked for bacterial growth in the next 72 h These plates were used as epiphytic controls for selectively picking endophytic colonies from the root samples (Bertani et al., 2016) To release the endophytic bacteria, the surface sterilized stems were macerated in 10 ml of PBS 1× solution by using sterile mortar and pestle, then the suspension was diluted with PBS 1ì Aliquotes of 50 àl from different dilution levels were plated on 1/5 TSA plates and incubated at room temperature for days Single colonies showing distinct morphology in comparison to the epiphytic controls were selected and purified again by streaking on 1/5TSA plates, then stored at −80°C in 18% glycerol/PBS for further experiments DNA extraction, PCR amplification, sequencing and phylogenetic analysis The bacterial genome DNA was extracted using the mini column Bacterial DNA Kit (Omega, USA) Amplification of 16S rRNA gene was performed with primer pairs 27F and 1492R (Weisburg et al., 1991) Prior to sequencing, the PCR products were purified with QIAquick PCR purification Kit (Qiagen) The sequencing was performed on an ABI 3110 Avant Applied Biosystems sequencer (ABI, USA) The 16S rDNA sequences were compared with related sequences available on the GenBank database by using the BLAST Search tool The alignment of sequences was performed by using CLUSTAL_X program, version 1.8 and a phylogenetic tree was reconstructed using the neighbor-joining method (Saitou, Nei, 1987) Topography of the reconstructed tree was evaluated by bootstrap analysis with 1000 replicates (Felsenstein,1985) Determining antagonistic activity against Dickeya zeae Dickeya zeae strain DZ2Q was cultured in Tryptic Soy Broth (TSB) (Himedia, India), shaken 160 rpm/min at 30°C for 24 h and used as testing pathogen TSA was sterilized, let to cool down to below 50°C, then pre-grown culture of Dz was added (1%, vol/vol), gently shaken and poured into Petri dishes To screen for the Dz antagonizing activity, the endophytic bacterial (EB) strains were cultured on TSA 1/2 plates Agar discs with EB colonies (diameter mm) were placed on the Petri dishes containing the Dz, the distances between the agar disks were ∼ cm (Jiménez-Esquilínet al., 2005) Crude extracts or extracted fractions obtained from HPLC were examined on Dz inhibitory activity by agar-well diffusion method (Magaldi et al., 2004) Aliquotes of 50 µl of sample were dripped into mm diameter wells created on agar plates containing Dz The plates were incubated for 24 h at 30 oC and Dz antagonism was determined by the size of the clearance zone (ΔD) formed around the agar wells according to the following equation: ΔD = D −d where: D is the diameter of the antibacterial zone (mm) d is diameter of the agar well (mm) Extraction of the Dz-inhibiting bioactive compound from strain VY81 Culture of strain VY81 was centrifuged at 4000 rpm/min for 20 minutes to remove cell biomass Four different solvents, i.e ethyl acetate, butanol, ethanol and n-hexane were tested for the extraction of the bioactive compounds The solvent (each of the four) was added to the culture broth in a 1: ratio and mixed (3 replicates) Afterward, the solvent and water phases were separated and the crude extracts were recovered from the solvent by evaporation in vacuum evaporator at 30°C In addition, ethanol was used to extract bioactive compounds from the cell biomass The ethanol and cell biomass mixture was centrifuged and the supernatant was subjected to vacuum evaporation The crude extracts were then re-dissolved in 15% acetonitrile (CH3CN) and tested for anti-Dz activity The experiment was carried out with culture broth of strain VY81 at different growth time (1-7 day) Purification and structural determination of the Dz-inhibiting bioactive compound from strain VY81 The crude extract was transferred to gel chromatography using C18 column (Agilent, USA) CH3CN/H2O was used as mobile phase in gradient elution of 15%, 30%, 45%, 60%, 80% and 100% (2× repetitions) The fractions were then tested for anti-Dz activity The fraction with the best activity was then analyzed by HPLC using Eclipse Plus C18 column (150 ì 4.6 mm; àm) (Agilent, USA), with CH 3CN concentration increased from 15 - 85% in 35 minutes at a rate of 1.2 ml/min Each fraction (1 min/fraction) was collected and tested for anti-Dz activity The peak corresponded to the fraction with the best inhibitory activity was collected, dried by lyophilization Chemical structure of the compound was analyzed by mass spectrometry (Agilent 1100 LC-MSD Trap, USA) and nuclear magnetic resonance spectroscopy (NMR) (Bruker AM500 FT-NMR Spectrometer, USA) RESULTS AND DISCUSSION Isolation of rice endophytic bacteria (EB) and screening for Dz-inhibiting activity Total 70 EB strains were isolated from different plant parts (root/stem/leaf) of the rice cultivar IR4625 planted in Long An in Spring-Summer season 2018 Strain VY81was isolated from surface sterilized stem, adjacent to the rot area The strain possessed prominent activity against Dz (Figure 1A) as shown in the screening experiment Strain VY81 had round (diameter of 2-3 mm), slightly convex, uniform, glossy, light yellow colonies when grown in TSA after 48 h at 30 °C Cells were of 1,4 – 2,2 × 0,5 – 0,6 µm in size, non-motile as observed under phase-contrast and scanning electron microscope (Figure 1B), Gram-negative bacillus Comparative analyses of 16S rRNA gene sequence indicated that strain VY81 belonged to the genus Burkholderia, most closely related to Burkholderia cepacia (99.78% sequence homology) (Figure 1C) The nearly full length of 16S rRNA gene sequence of strain VY81 was deposited at GenBank (NCBI) underaccession number MW056196 Figure Antibacterial activity against Dz, cell morphology and taxonomic position of the rice endophytic bacterium VY81 (A) Dz inhibition zone (agar plug diffusion method); (B) Cell morphology under scanning electron microscope (SEM), magnification of 10,000×; (C) Taxonomic position based on 16S rDNA sequence comparison of strain VY81 with related species Burkholderia spp have been reported to be plant EB, providing various benefits to the host plants such as modulating growth and stress, related phytohormones and nitrogen fixation (Doty et al., 2016) In addition, Burkholderia spp that grow in the rhizosphere are also capable of decomposing toxic pollutants and/or inhibiting phytopathogens via production of variety of secondary metabolites such as antibiotics, enzymes (SuárezMoreno, 2012) Potential applications of Burkholderia spp in agriculture to promote plant growth and control pathogens have been reported recently (Paungfoo-Lonhienne et al., 2014; Bernabeu et al., 2015) Extraction and purification of Dz-inhibiting bioactive compound All crude extracts from culture broth of strain VY81 using three solvents n-hexane, ethyl acetate and butanol showed antagonistic activity against Dz (Figure 2A) It is shown that ethyl acetate and butanol had similar extraction efficiency, whereas n-hexane produced a crude extract with ∼ 40% lower antagonistic activity The crude extract from cell biomass of strain VY81 with ethanol had low Dz-inhibitory effect, at the same level of the culture broth extracted by n-hexan, indicating that the target compound accumulated at higher concentration extracellular in the culture broth Ethyl acetate was selected to perform extraction of the Dz-inhibiting compound since it has higher volatility than butanol, i.e is more convenient of being removed by evaporation.Tests of crude extracts from culture broths of different ages also indicated that 48 hours was the best time for harvesting the target compound from culture of strain VY81 (Figure 2B) Figure Extraction of bioactive compound from VY81 strain (A) The extraction efficiency of the different solvents (B) Anti-Dz activity at different culture times Strain VY81 was grown in TSB for 48 hours and culture broth was treated with ethyl acetate, the crude extract was then recovered after evaporating the solvent and redissolved in 15% ACN The first purification step was performed using C18 gel chromatography column, eluted with different concentrations of CH3CN (15%, 30%, 45%, 60 %, 80% and 100%) The highest Dz-inhibiting activity was observed at the 60% CH3CN fraction This fraction was then selected for the next purification step using C18 HPLC The highest Dz-inhibiting activity was observed at fraction 17, therefore, the single HPLC peak at 16.2 - 16.8 minutes was collected This experiment was repeated 100 times to obtain 20 mg pure compound for further analyses Determination of chemical structure of the Dz-inhibiting compound from strain VY81 Compared with previously published researches, Dz-inhibiting compound produced by strain VY81 has a characteristic UV absorption spectrum (Figure 3A), similar to that of compound 2-(2-heptenyl)-3-methyl-4-quinolinol (chemical formula C17H21ON) (Hashimoto, Hattori, 1967) (Figure 3B) The double branching in the 320 - 340 nm region is characteristic for the 4-quinolinol derivatives (Hashimoto, Hattori, 1967) In 1990, Roitman et al reported on compounds 2-(2-heptenyl)-3-methyl-4(1H)-quinolone with UV spectrum similar to that of Hashimoto and Hattori (Roitmanet al., 1990) Figure UV absorption spectra of anti-Dz bioactive compound from VY81 strain (A) and 2- (2heptenyl) -3-methyl-4-quinolinol (B) (Hashimoto & Hattori, 1967) The purified target compound was lyophilized to remove the solvent and was then analyzed for chemical structure by electrospray ionisation mass spectrometry (ESI-MS) and 1H-NMR and 13 C-NMR Mass spectrometry analysis showed that the Dz-inhibiting compound produced by strain VY81 had m/z of 255.8 [M + H] + Meanwhile, the nuclear magnetic resonance spectrum analysis showed that the compound had chemical formula C17H21ON and chemical name 2- (2-heptenyl) -3-methyl-4 (1H) -quinolone (HMQ), similar to the compound (Figure 4B) reported by Roitman et al (Roitman et al., 1990) Figure Chemical structure of (A) 2-(2-heptenyl)-3-methyl-4-quinolinol (Hashimoto & Hattori, 1967) and (B) 2-(2-heptenyl)-3-methyl-4(1H)-quinolone (Roitman et al., 1990) The compound 2-(2-heptenyl)-3-methyl-4-quinolinol was extracted for the first time with acetone from Pseudomonas pyrrocinia in 1967 Mass spectrometry, UV spectroscopy and NMR spectroscopy showed that this compound has a molecular weight of 255, chemical formula C17H21ON, chemical structure as shown in Figure 4A (Hashimoto, Hattori, 1967) The results of extraction from the culture of Pseudomonas cepacia strain RB425 (later Burkholderia cepacia RB25) isolated from lettuce roots by chloroform/ethyl acetate/benzene also showed similar results (Homma et al., 1989) This alkaloid compound exhibits antagonistic activity against many fungal and bacterial plant pathogens, including Pyricufaria oryzae, Rhizoctonia solani, Pythium ultimum, Furasium oxysporum, Verticillium dahliae, Gaeumannomyces graminis, Cochliobolus miyabeanus (Homma et al., 1989) This compound is a derivative of compound 2-(2-heptenyl)-3methyl-4(1H)-quinolone extracted from P cepacia LT4.12-W (now is B cepacia LT4.12W) isolated from apple leaves using acetone and methanol solvents (Roitman et al., 1990) The crude extract was analyzed by antiphase HPLC C18 column with the mobile phase CH3CN/H2O (3:2, v/v) The obtained bioactive compound was purified by HPLC cyanosilica column with the mobile phase CHCl/hexane (1:1) The resulting compound is colorless crystal, 13C-NMR, δ10.6 (C3-Me), 13.9(C7’), 22.2 (C6’), 31.3 (C5’), 32.2 (C4’), 35.6 (Cl’), 115.6 (C3), 117.3 (C8), 123.0 (C6), 123.3 (C3’), 123.6 (ClO), 125.8 (C5), 131.1 (C7), 135.5 (C2’), 139.2 (C9), 147.9 (C2), 178.0 (C4) The UV spectrum was similar to that of Hashimoto and Hattori, 1967 (Figure 3B) (Roitman et al., 1990) The physical constants of the bioactive compound produced by VY81 strain were similar to that of this compound, so the active ingredient extracted in this study was 2-(2-heptenyl) -3-methyl4(1H)-quinolone HMQ belongs to the quinoline alkaloids group of heterocyclic aromatic compounds with many important biological activities Many alkaloid quinoline compounds have been discovered from natural sources, including several families of the plant kingdom, as well as from animals and microorganisms (Kshirsagar et al., 2015) Quinine was the first quinoline alkaloid compound isolated from the quinquina phloem (Cinchona spp.) in 1820 and used as a substitute for crude phloem in malaria treatment (Shang et al., 2018) Quinoline alkaloids containing pyrrole extracted from Streptomyces spp were inhibitory to tumors and cancer cell lines Quinoline alkaloids and their derivatives were widely used in medicine and agriculture, notably anti-malarial drugs (Quinine, Quinidine, Chloroquine, Mefloquine, etc.), antiviral (Saquinavir, etc.), anti-cancer (Camptothecin, Irinotecan, Topotecan, Gefitinib, etc.), antipsychotic drugs (Aripiprazole, Brexpiprazole, etc.), antiglaucoma (Cartiolol) and cardiotonic (Vesnarin) (Selvan et al., 2011; Afzal et al., 2015; Tiwaryet al., 2015; Patel et al., 2017) The endophytic bacterium Burkholderia cepacia VY81 isolated in this study is evidently capable of biosynthesizing quinolone, which effectively inhibits Dickeyazeae The EB strain and its quinolone-derivative compound are supposed to have potential applications in biological control of the rice foot rot disease caused by Dz Using natural compounds that antagonize phytopathogens has become an indispensable trend in sustainable agricultural development The results of this study also showed that plant endophytes are potential sources to search for bioactive compoundssupporting organic agriculture CONCLUSION Endophytic bacterial strain VY81 isolated from rice stem exhibited high antagonistic activity against Dickeya zeae, the phytopathogen causing foot rot disease Comparative analyses of 16S rRNA gene sequences allowed classifying strain VY81 into the genus Burkholderia, 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trường, trường Đại học Phenikaa Trung tâm nghiên cứu chuyển giao công nghệ, Viện Hàn lâm Khoa học Công nghệ Việt Nam Khoa Sinh học, trường Đại học Khoa học Tự nhiên, Đại học Quốc gia Hà Nội TÓM TẮT Hệ vi khuẩn nội sinh thực vật chứng minh mang nhiều đặc tính có lợi cho chủ nhìn nhận probiotics thực vật Nhiều chủng vi khuẩn nội sinh cơng bố có hoạt tính kháng tác nhân gây bệnh trồng, bao gồm vi khuẩn, nấm tuyến trùng Sử dụng vi khuẩn nội sinh cho mục đích kiểm sốt sinh học có tiềm ứng dụng lớn, góp phần giảm sử dụng hóa chất nông nghiệp tiến tới phát triển nồng nghiệp bền vững Trong nghiên cứu này, vi khuẩn nội sinh phân lập từ giống lúa IR4625 trồng Long An sử dụng để sàng lọc tìm kiếm chủng có khả kháng vi khuẩn Dickeya zeae (Dz) gây bệnh thối gốc lúa Các mẫu lúa có biểu bệnh rõ rệt phần gốc có màu nâu sẫm, mùi thối nhiễm vi khuẩn Chủng VY81 phân lập từ phần thân lúa gần với điểm thối vi khuẩn sau khử trùng bề mặt Kết nghiên cứu hoạt tính đối kháng Dz sử dụng dịch chiết thô chủng VY81 cho thấy chủng có khả ức chế Dz cao với đường kính vịng kháng khuẩn 14,25 mm ± 1,06 Chủng VY81 sinh hoạt chất kháng Dz nồng độ cao sau 48 h sinh trưởng môi trường TSB, hoạt tính kháng tìm thấy chủ yếu dịch canh trường, phần nhỏ sinh khối tế bào Hoạt chất kháng Dz chủng VY81 tiết vào dịch canh trường tinh HPLC xác định cấu trúc hóa học qua phân tích phổ khối cộng hưởng từ hạt nhân Kết hợp thuộc nhóm quinoline alkaloid, có cơng thức hố học C17H21ON danh pháp hố học 2-(2-heptenyl)-3-methyl-4(1H)quinolone So sánh trình tự gen 16S rDNA cho phép xếp chủng VY81 vào chi Burkholderia, loài gần gũi B cepacia (99,77% tương đồng trình tự) Trình tự 16S rDNA chủng VY81 đăng ký GenBank với mã số MW056196 Chủng VY81 hoạt chất quinolone có tiềm ứng dụng để phát triển sản phẩm phòng chống bệnh thối gốc vi khuẩn Dickeya zeae gây trồng Từ khóa:Bệnh thối gốc lúa, Burkholderia cepacia, Dickyea zeae, kiểm soát sinh học, quinoline, vi khuẩn nội sinh lúa Author for correspondence: Tel: +84-972523466; E-mail: dthangimbt@gmail.com ... ? ?Nghiên cứu vi khuẩn nội sinh lúa nhằm ứng dụng phòng trị bệnh thối rễ Dickeya zeae gây ra? ?? để tìm lồi vi khuẩn nội sinh hoạt chất sinh học chúng đối kháng vi khuẩn gây bệnh Dickeya zeae nhằm ứng dụng. .. GIA HÀ NỘI TRƯỜNG ĐẠI HỌC KHOA HỌC TỰ NHIÊN - NGUYỄN DUY TỚI NGHIÊN CỨU VI KHUẨN NỘI SINH LÚA NHẰM ỨNG DỤNG TRONG PHÒNG TRỊ BỆNH THỐI RỄ DO Dickeya zeae GÂY RA Chuyên ngành: Vi sinh vật... 10 Chuyên ngành Vi sinh vật học Mục tiêu đề tài • Phân lập tuyển chọn chủng vi khuẩn nội sinh lúa có hoạt tính đối kháng vi khuẩn Dickeya zeae gây bệnh thối thân /rễ lúa • Nghiên cứu, xác định hoạt