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MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY NGUYEN ANH HUY ISOLATION AND SELECTION OF SALT TOLERANT BACTERIA WHICH HAVE NITROGEN FIXING CAPACITY AND INDOLE ACETIC ACID SYNTHESIS ISOLATED FROM SOIL OF RICE – SHRIMP PRODUCTION IN BAC LIEU, SOC TRANG AND KIEN GIANG PROVINCES Major: Biotechnology PHD THESIS SUMMARY Can Tho, 2019 MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY NGUYEN ANH HUY ISOLATION AND SELECTION OF SALT TOLERANT BACTERIA WHICH HAVE NITROGEN FIXING CAPACITY AND INDOLE ACETIC ACID SYNTHESIS ISOLATED FROM SOIL OF RICE – SHRIMP PRODUCTION IN BAC LIEU, SOC TRANG AND KIEN GIANG PROVINCES Major: Biotechnology Coded: 94 20 201 PHD THESIS SUMMARY Supervisor Ass Prof Dr NGUYEN HUU HIEP Can Tho, 2019 The thesis research was completed in: Biotechnology Research and Development Institute, Can Tho University Supervisor: Ass Prof Dr Nguyen Huu Hiep Reviewer 1: Reviewer 2: The thesis will be available for reference in: Learning Resource Center – Can Tho University Vietnamese National Library CHAPTER GENERAL INFORMATION OF THE THESIS 1.1 Necessity of thesis In the context of climate change and global warming, salinity intrusion is one of the urgent problems of the agricultural sector as it directly affects the productivity and quality of agricultural products The Mekong Delta is the main rice-producing region of the country, which is also heavily impacted by climate change When the soil is salty, the nutrient and water absorption capacity of rice is limited In addition, in order to protect the environment for shrimp farming, people restrict the use of chemical fertilizers and pesticides As a result, the rice crop yields are very low Bacteria in the root zone are capable of fixing nitrogen and IAA synthesis to stimulate the growth and development of the root system to increase the uptake of nutrients from the soil, while providing protein for rice Therefore, the topic of "Isolation and selection of salinity tolerant bacteria that can fix nitrogen, IAA synthesis on rice-shrimp farming model in Bac Lieu, Soc Trang and Kien Giang" was conducted wrongly selected lines Local bacteria have the ability to fix nitrogen and IAA synthesis with high content At the same time, it is effective in reducing the amount of chemical fertilizers and increasing the productivity of rice cultivated on saline soils 1.2 Objectives Salinity-tolerant bacterial strains that are able to fix nitrogen and free IAA synthase in farmland under the riceshrimp farming model 1.3 Object and scope of research Salinity-tolerant bacterial strains that are able to fix nitrogen and free IAA synthase in farmland under the riceshrimp farming model Using non-protein Burk, supplemented with 10 mu salt isolate free-living bacteria strains in rice-rooted land with nitrogen fixation capacity and effective IAA biosynthesis on rice in the rice- in three provinces Bac Lieu, Soc Trang and Kien Giang The scope of the study only investigated the ability of nitrogen fixation and IAA synthesis, did not investigate the phosphorus solubility of bacteria 1.5 New contributions of the thesis The main objective of the study was the isolation, selection and identification of free-living salinity tolerant strains in rice-root soils planted on saline soils capable of nitrogen fixation and potential IAA synthesis Production of micro fertilizers used in the rice-shrimp farming model 1.4 Scientific significance and practical significance The topic has identified 216 salinity tolerant lines that have identified the morphology and biochemical character as well as the ability of nitrogen fixation and IAA synthesis Of these, 35 were sequenced by the 16S rDNA gene This is the source for further research as well as a supplementary source of reference textbooks CHAPTER MATERIALS AND METHODS 2.1 Research contents Isolation of salinity tolerant strains capable of nitrogen fixation and IAA synthesis, free survival in rice root zone, rice and shrimp production in Bac Lieu, Soc Trang and Kien Giang Investigation of morphological characteristics and biochemical characteristics of isolated strains Determination of nitrogen fixation capacity and IAA synthesis of isolated strains Identification of highly efficient strains by 16S rDNA gene sequencing Test to evaluate the effectiveness of bacterial strains grown under laboratory conditions, in net houses and in the field 2.2 Research methodology 2.2.1 Isolation and examination of the nitrogen fixing capability of rice rhizosphere bacteria Soil samples and cultural media Rice rhizosphere soil samples were collected from provinces of the Mekong Delta including Bac Lieu, Soc Trang and Kien Giang Each provinces (city) randomly selected 3-5 districts, in each districts chose 1-3 communes, each communes selected 1-5 rice cultivation fields Five hills of rice (at 1-2 months old) were collected in each fields Lightly separating muddy soil around the rice roots into plastic bags, labeled (approximately kg/sample), and brought to the laboratory for isolation Cultural media: Group of rice rhizosphere bacteria was isolated on Burk medium without nitrogen mineral (Park et al., 2005) including Sucrose (10 g/l), KH2PO4 (0.41 g/l), K2HPO4 (0.52 g/l), Na2SO4 (0.05 g/l), CaCl2 (0.2 g/l), MgSO4 7H2O (0.1 g/l), FeSO4.7H2O (0.005 g/l), NaMoO4.2H2O (0.0025 g/l), Agar (18 g/l) Isolation of bacteria Weighed 10 g of soil samples, added 90 ml of sterile distilled water, put into sterile flasks, samples were stirred by magnetic stirrer for hours, let stand for hour, then diluted into decimal range 10-1, 10-2, 10-3… Pipetted 50 μl of samples (in each concentration) using a micropipette, dripped on agar plates containing Burk medium without nitrogen mineral (each concentration plates) Using a sterile glass rod spread the sample drops on the surface of medium, covered the plate and standard for a few minutes then turning the disk down, and incubated cultural plates at 30oC in incubator Chose the separate colonies with different colors, shapes and sizes, sub cultured many times on isolation medium by streak plate method, and examined under a microscope to determine the pure isolates of bacteria Checking the nitrogen-fixing ability of the bacterial strains Nitrogen-fixing bacteria can thrive on cultural medium without nitrogen mineral due to their ability to synthesize ammonium from atmosphere nitrogen The bacterial strains with capability of growing well were selected and sub cultured in liquid Burk medium to measure levels of ammonium in cultural medium by Indophenol Blue method (Page et al., 1982) Checking the IAA synthesis ability of the bacterial strains Prepare the original bacteria Multiply the block of lines in the Falcon tube containing mL of Burk's non-nitrogenous liquid medium Put on a shaker, shake 60 rpm, 48 hours, using black plastic bag to shine during the culture Transfer mL of proliferating bacteria to a Falcon tube containing 10 mL of non-protein liquid nitrogen supplemented with 100 mg / L L-Tryptophan, fed on a shaker Quantify the ability of IAA synthesis of bacteria on days 2, 4, Repeat three times for each bacterial strain Construction of the IAA benchmark Calculate 0.0016g of IAA in 10 mL phosphate buffer solution with 160 μg / mL IAA Dilute to concentrations of 2.5, 5, 10, 20, 30, 40, 80 μg / mL for test tubes Prepare test tubes and numbered from to Tube contains mL of reagent and mL of phosphate buffer The remaining tubes, each containing mL of reagent and mL of the standard diluted IAA solution, correspond to concentrations of 2.5, 5, 10, 20, 30, 40, 80 μg/mL Figure 1: Color reaction of test tubes to IAA standard The bacterium should investigate the ability of IAA synthesized after nitrification to bring the centrifuge at 4ºC at 13,000 rpm and 10 minutes to remove the cells Prepare test tubes for each sample Vacuum mL of the inner solution after centrifugation into the test tube, containing mL Salkowski reagent, bring the vortex To stabilize at room temperature for 15 - 30 minutes Measure the absorption intensity at 530 nm From the results of OD530nm measure of potential input into the equation, the inferred IAA synthesis ability of each strain of bacteria to be investigated Identification of bacterial strains capable of synthesizing 𝐍𝐇𝟒+ and IAA with high content by 16S rDNA gene sequencing Sequencing the PCR products of 16S rDNA gene using universal primers 27F and 1495R at Biotechnology Research and Development Institute, Can Tho University and the Macrogen company (South Korea), and compared with the NCBI gene bank to determine the genetic relationships of selected bacterial strains with the corresponding species in gene bank Evaluation of the nitrogen-fixing effect of promising bacterial strains for rice grown Assay for effective nitrogen fixation on rice under laboratory conditions based on the (Ngo Thanh Phong (2013) and Nguyen Thi Pha (2015) to select bacterial strains that can fix nitrogen Assay for effective fixation of nitrogen to rice under potted conditions, irrigated with 7NaCl: 2CaCl2: 1Na2SO4 (Predeepa and Ravindran, 2010) Selection of bacterial strains with N-fixing ability of rice cultivated in the field, experiments were arranged on rice-shrimp land in Gia Hoa commune, My Xuyen district, Soc Trang province Vo Van Be et al (2013), in the field of Soil Science, Faculty of Agriculture and Applied Biology, Cantho University fertilizer formula and number of times recommended by the Center for Plant Breeding in Soc Trang CHAPTER RESULTS AND DISCUSSIONS Results of land sampling from land for shrimp rice production The soil samples of the saline zone were collected in two batches, the first one was from October 2014 to March 2015 in two provinces of Bac Lieu and Soc Trang, the second was collected in February 2016 in Kien Giang The result obtained 65 acres of land in 65 rice fields under the rice - shrimp model Bac Lieu collected 12 acres in Phuoc Long and Hong Dan districts Soc Trang collected 36 acres in districts of My Xuyen, Tran De, Long Phu Kien Giang province collected 17 acres in An Minh and Hon Dat districts Results of isolation of free-living saline bacteria in rice-shrimp production land with nitrogen fixation ability From 65 soil samples of salinized rice roots in Bac Lieu, Soc Trang and Kien Giang provinces The subject has isolated 216 bacterial strains From the samples of rice fields infected with salty water in Bac Lieu province, 53 strains of bacteria were isolated From the soil sample of Soc Trang province, 78 bacterial strains were isolated Kien Giang soil samples were isolated from 85 strains 78/216 85/216 53/216 Sóc Trăng Bạc Liêu Kiên Giang Figure 2: Number of bacterial isolates in each province Colony characteristics: Amphibians have two forms, round (158/216, 73.1%) and irregular (58/216, 26.9%); Colors are available in two colors, mostly white (189/216, 87.5%) and yellow; Two types of cardboard, 80.6% of original cardboard and 19.4% of cardboard; There are three forms, tissue (80.1%), long (19%), flat (0.93%) Figure 3: Colony of Burkholderia sp PL9 (A) and Acinetobacter sp GH1-1 (B) on free-Nitrogen Burk added substitute salt 10‰ Bacterial cell characteristics: Short-rod cells (64.8%), rods long (20.4%), spherical cells (7.9%), bacterium (6.9%); mobility, most of the strains are capable of movement (69%), non-moving (31%); Gram, the Gramnegative and Gram-negative strains are approximately equal Ngày 34 Ngày 1,00-1,99 µg/mL 11 2,00-2,99 µg/mL