Phân lập và tuyển chọn vi khuẩn chịu mặn có khả năng cố định đạm, tổng hợp INDOLE ACETIC ACID trên mô hình canh tác lúa – tôm ở bạc liêu, sóc trăng và kiên giang tt tiếng anh

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 SYNTHES

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:

1 Learning Resource Center – Can Tho University

2 Vietnamese National Library

CHAPTER 1 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 rice-shrimp 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 rice-shrimp 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 2 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 3

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 1 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 2 hours, let stand for 1 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 3 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 3 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 1

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, 6 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 7 test tubes Prepare 8 test tubes and numbered from 0 to 7 Tube 0 contains 2 mL of reagent and 1 mL of phosphate buffer The remaining tubes, each containing 2 mL of reagent and 1 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 3 test tubes for each sample Vacuum 1 mL

of the inner solution after centrifugation into the test tube, containing 2 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 2 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 3 RESULTS AND DISCUSSIONS

1 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 3 districts of My Xuyen, Tran De, Long Phu Kien Giang province collected 17 acres in An Minh and Hon Dat districts

2 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

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 Gram-negative and Gram-Gram-negative strains are approximately equal

78/216

53/216 85/216

Sóc Trăng Bạc Liêu Kiên Giang

3 The ability to synthesize 𝐍𝐇𝟒+ of bacterial isolates

Two hundred and sixteen isolated strains of bacteria were able to fix nitrogen, the concentrations of synthetic bacterial protein changed over the days of survey, day 2, day 4 and day 6 The prevailing trend (51.4%) was increased on Day 4 and decreased on Day 6 On Day 2, there were 15/216 (7%) bacterial strains capable of synthesizing NH4+ at concentrations of 3.00 - 4.32 μg/mL; Day 4 contained 21/216 (10%) bacterial strains capable of synthesizing NH4+ with concentrations of 3.00 - 5.33 μg/mL; At day 6, none of the bacterial strains synthesized with NH4+ ≥ 3.00 μg/mL had only 11/216 (5%) bacterial strains capable of synthesizing NH4+ , 95 μg/mL

Figure 4: Concentration of NH4+ of 216 bacterial strains

over 2, 4, 6 days of survey

4 IAA synthesis capability of isolated strains two hundred and sixteen bacterial isolates are capable of IAA synthesis

The IAA activity of isolates of salinity-tolerant bacteria tended to decrease in days 2, 4 and 6 (99/216 bacterial strains, 45.8%) On day 2, there were 11/216

160

21 16 28

151

34 171

0

20

40

60

80

100

120

140

160

180

(5.1%) strains capable of synthesizing IAA from 50.00 to 66.53 μg/mL; On day 4, strains of bacteria tended to reduce IAA synthesis, with 8/216 (3.7%) of the IAA synthetic strains reaching 50.00 - 58.98 μg/mL; At day 6, the strains tended to further reduce IAA levels, with 7/216 (18.5%) of the IAA bacterial strains reaching 30.00 - 39.99 μg/mL

Figure 5: Concentration of IAA of 216 bacterial strains

over 2, 4, 6 days of survey

5 Nitrogen fixation of 4 bacterial strains was highly effective for rice at plating stage, at salinity of 0 ‰, 5‰ and 10‰

Four strains of PLA (PL2, PL9, GH1-1, L1) were able to fix nitrogen and effective IAA synthesis on rice at plating stage Solution of salted solution (7NaCl: 3CaCl2) with salinity of 0‰, 5‰ and 10‰ The results showed that the ability of ammonium synthesis in all four bacterial strains decreased at salinity of 5 and 10‰ Specifically, the PL9 and GH1-1 strains were able to synthesize ammonium in the medium supplemented with salt 5‰, (1,34 and 1,12 μg/mL) Thus, although all four isolates were cultured on cultured media with a salinity of 10‰, the ammonium content in the culture media was reduced

11

86

8

95

0 7

40 37 132

0

20

40

60

80

100

120

140

Hàm lượng IAA µg/mL