Phân lập, tuyển chọn và định danh vi khuẩn liên kết với thực vật (plant associated bacteria) ở lúa, khoai trồng trên đất phèn ở đồng bằng sông cửu long tt tiếng anh

MINISTRY OF EDUCATION AND TRAINING
CAN THO UNIVERSITY

DOCTORAL THESIS SUMMARY
Specialized: Soil Science
Code: 9620103
LY NGOC THANH XUAN

ISOLATION, SELECTION AND IDENTIFICATION
PLANT ASSOCIATED BACTERIA AT RICE,
SWEET POTATO CULTIVATED ON
ACID SULPHATE SOILS IN
THE MEKONG DELTA

Can Tho, 2019


THE Ph.D. THESIS WAS COMPLETED
AT CAN THO UNIVERSITY

Advisor: Assoc. Prof. Dr. TRAN VAN DUNG
Prof. Dr. NGO NGOC HUNG

The doctoral thesis was evaluated by The Board of Examiners at
basic level.
Meeting at: Meeting room 3, 2nd floor, administrative house, Can
Tho University.
At 14.00 p.m, date 22/6/2018.

Reviewer 1: Dr. LUU HONG MAN.
Reviewer2: Dr. CAO VAN PHUNG.

You can find thesis at the library:
Learning Resource Center, Can Tho University.
National Library of Vietnam.


LIST OF PUBLICTION
1. Ly Ngoc Thanh Xuan, Trinh Quang Khuong, Le Van Dang, Tran
Van Dung và Ngo Ngoc Hung, 2016. Applcation of
plantassociated bacteria Burkholderia vietnamiensis on the
growth and yield of rice crops cultivated on acid sulphate soil
in the Mekong Delta. Can Tho University Journal of Science,
44b: 1-8.
2. Ly Ngoc Thanh Xuan, Tran Van Dung, Ngo Ngoc Hung,Cao
Ngoc Diep, 2016.Isolation and characterization of rice
endophytic bacteria in acid sulphate soil of Mekong delta,
Vietnam. World journal of Pharmacy and Pharmaceutical
sciences,5 (8): 301-317.
3. Ly Ngoc Thanh Xuan, Tran Van Dung, Ngo Ngoc Hung,Cao
Ngoc Diep, 2016.Isolation and characterization of rhizospheric
bacteria in rice (oryza sativa l.) cultivated on acid sulphate
soils of the Mekong delta, Vietnam. World journal of
Pharmacy and Pharmaceutical sciences, 5 (9): 343-358.
4. Ly Ngoc Thanh Xuan, Tran Van Dung, Ngo Ngoc Hung,Cao
Ngoc Diep, 2017. Isolation and characterization of endophytic
and rhizopheric bacteria associated sweet-potato plants
cultivated on soils of the Mekong delta, Vietnam. World
journal of Pharmacy and Pharmaceutical sciences, 6 (1): 129149.
5. Ly Ngoc Thanh Xuan, Le Van Dang, Tran Van Dung and Ngo
Ngoc Hung, 2018.Applcation of plantassociated bacteria on

the yield of sweet potato cultivated on acid sulphate soil in the
Mekong Delta. Science and Technology Journal of Agriculture
and Rural Development, Ministry of Agriculture and Rural
Development, Vietnam, 7: 93-103.

1


INTRODUCTION
1. Rationale and background
Nitrogen (N) is the most essential nutrient to promote rapid plant
growth and grain yield; thereby, high amounts of chemical N
fertilizers have been applied to gain high crop yield in agricultural
ecosystems for intensive fertilization practices (Galloway et al.,
2004), especially in low-fertility soils like ASS as an example. This
issue faces not only environmental problems as greenhouse gas
emission but also microbial communities as changes of N2-fixer
growth and activity (Reardon et al., 2014; Tang et al., 2017).
Therefore, a biological nitrogen source should be altered to tackle
with this problem and make a use of natural resources. Specifically,
biological nitrogen fixation (BNF) is one of the important natural
processes to fix N2 from the atmosphere into bioavailable NH4+ in the
soil by nitrogenase enzyme, which is an important source of nitrogen
in agricultural terrestrial systems (Peoples and Craswell, 1992;
Kennedy and Islam, 2001). Normally, insufficient phosphorus (P)for
rice has been observed in ASS due to immobilized P asits
precipitation with free aluminum and iron ions to form aluminum
phosphate (AlPO4) and iron phosphate (FePO4) (Margenot et al.
2017); and this leads toless available forms of P for plants (Rengel
and Marschner 2005) and altered the soil microbial community

structure (Ragot et al. 2016; Soman et al. 2017). Hence, several tools
to resolve this problem include chemical, physical and biological
approaches. Rice cultivation in actual ASS faced with toxicity of
toxicity (Högfors-Rönnholm et al. 2018) and acidic environment that
cause low yield (Johnston et al. 2016). The ASS can be reclaimed by
physical and chemical techniques although there are many
constraints of both systems such as high costs, water shortage and
Cd contamination derived from phosphorus fertilizers (Chaitanya et
al. 2017; Shamshuddin et al. 2017). However, these problems can be
tackled by the endophytic bacteria and rhizobacterial strains because
they possesses the ability of N2-fixation, P-solubilization from P-Al,
and P-Fe sources. Moreover, the efficacy of microorganism is
depended on interaction between bacteria and host (Patnailk, 1994).
1


Regarding above information, to achieve the sustainable agricultural
system for solving the serious problems of rice cultivation on ASS,
isolation, selection and application of endophytic bacteria and
rhizobacterial strains from rice, sweet potato and their soils are
necessary.
2. Objectives
(i) Isolation of endophytic and rhizospheric bacterial strains
associated with sweet potato and rice for abilities of nitrogen fixing,
phosphate-solubilizing;
(ii) Selection of promising nitrogen fixing, phosphatesolubilizing bacterial strains for improvement of sweet potato and
rice growth and yield in the greenhouse and field conditions;
(iii) Suggestion of promised 2-3 strains to apply on acid
sulfate soil.
3. Novelty of research

The without nitrogen fertilizer application decreased rice
yield on acid sulfate soil in Long My – Hau Giang, Hon Dat – Kien
Giang and Hong Dan – Bac Lieu, but the application of both 60 kg N
ha-1 and Burkholderia vietnamiensis X1 resulted in higher rice yield
compared to control treatment (90N-60P-30K) on acid sulfate soil in
Hon Dat – Kien Giang. Similarly, the applied B. vietnamiensis X3
increased productivity in Long My – Hau Giang, and Hong Dan –
Bac Lieu. However, without phosphorus fertilizer application have
not been improved yield by application of B. vietnamiensis X1 và B.
vietnamiensis X3.
The bacterium Burkholderia acidipaludis X5 has high
nitrogen fixation capability compared to those in the 2 other bacteria
through increasednumber oftubersand yield of sweet potato.
Applying 60kgN/hain combination with incubation ofbacteria
Burkholderia acidipaludis X5 showed that the tuber number,
tuberlength, tuber diameter and sweet potatoyield were equivalent
with applying 90 kg N/ha. Incubation with bacteria Burkholderia
acidipaludis X5 could save30% of nitrogenfertilizersforsweet potato.
2


4. Outline of thesis content
The thesis included 103 pages with 5 chapters. Chapter 1:
Introduction (Pages: 1-5); Chapter 2: Literature review (Pages: 635); Chapter 3: Materials and methods (Pages: 36-58); Chapter 4:
Results and discussion (Pages: 59-102); Chapter 5: Conclusions and
recommendations (Pages: 103-104).
CONTENT OF THESIS
Chapter 1: Literature review
Rice (Oryza sativa L.) is one of the most important crops in the
world and paddy soil comprise the largest anthropogenic wetland on

earth (Kogel-Knabner et al. 2010) and the main dietary component
of 20% of the world’s population (IRRI, 2010)(Seck et al., 2012);
feeding more than 50% of the world’s population (Gyaneshwar et al.,
2001). In the next three decades, the world will need to produce
about 60% more rice than today’s global production to feed the extra
billion people (Ladha and Reddy, 2003). Increases in the demand for
rice, as a result of an increase in population, creates the need to
improve rice productivity and one of the most important factors for
high yields of rice production are chemicals fertilizers and
pesticides, which may cause environmental pollution and negatively
influence human health.
Sweet-potato (Ipomoea batatas (L.) Lam.), a dicotyledonous plant
belongs to the Convolvulaceae family, is a subsistence crop which
store starch in their roots and form tuberous roots. It has a huge
economic and social importance in developing countries (Souza and
Lorenzi, 2008) with over 95% of the global sweet potato crop which
is produced in developing countries (Reiter et al., 2003). It also is
resilient, easily propagated crop, growing well in infertile and
nitrogen (N) poor soils (Khan and Doty, 2009). Besides rice and fruit
plants, sweet potato plant also is cultivated popularly on alluvial soil
in the dry season as crop for food and exporting in the Mekong
Delta, Vietnam. Sweet potato area occupied 21,500 ha (9.3% sweet
potato area in Vietnam) with yield over 20 tons/ha in the Mekong
Delta (Agricultural Statastic in 2010).
3


In the kinds of chemical fertilizers, substances composed of
known quantities of nitrogen, phosphorus and potassium, keep an
important role in the growth and yield of crops as high-yielding rice.

Nitrogen (N) is one of the most important nutrients in plants and is a
limiting in plant growth and development and available phosphorus
(P) deficiencies in many parts of the world (Fageria et al. 2011) also
is the limiting factor for rice growth and development especially root
development, poor flowering and lack of seed etc., consequently
causing degradation in quality and quantity (Ji et al. 2014).
Soil is replete with microscopic life forms including bacteria,
fungi, actinomycetes, protozoa and algae. Of these different
microprganisms, bacteria are by far the most common (i.e., 95%). It
has been known for some time that the soil hosts a large number of
bacteria (often around 108 to 109 cells per gram of soil) and that the
number of culturable bacterial cells in soils is generally only about
1% of the total number of cells present (Schoenborn et al. 2004).
They are involved in various biotic activities of the soil ecosystem to
meke it dynamic for nutrient turn over and sustainable for crop
production (Ahemad et al., 2009). Most plants depend on soil, but
plants and their associated microorganisms also play a crucial role in
the formation or modification of soil (Pate and Verboom, 2009;
Taylor et al., 2009) and microorganisms present in the rhizosphere
play important roles in the growth and in the ecological fitness of
their plant host (Buee et al., 2009). Microbial interactions with roots
may involve either endophytic or free living microorganisms and can
be symbiotic, associative or casual in nature; beneficial
microorganisms include N2-fixing bacteria in association with
legumes and interaction of roots with mycorrhizal fungi and
phosphate-solubilizing microorganisms in relation to plant P uptake,
enhancement of root growth (Raaijmakers et al, 2009).Therefore, the
plant growth promoting rhizobacteria (PGPR), are characterized by
the following inherent distinctiveness’s: (i) they must be proficient to
colonize the root surface (ii) they must survive, multiply and

compete with other microbiota, at least for the time needed to
express their plant growth protion/protection activities, and (iii) they
must promote plant growth (Kloepper, 1994).
4


Endophytic bacteria are microorganisms that live in plant tissues
and they may be responsible for the supply of biologically fixed
nitrogen to their host plant (Boddey et al., 2005). Endophytes also
promote plant growth by a number of similar mechanisms as
phosphate solubilization activity (Waklin et al., 2004), indole acetic
acid production (Lee et al., 2004) and the production of a
siderophores (Costa and Lopez, 1994). Recently, the potential for
microbes to increases nutrient availability and to enhance crop
growth has garnered the attention of the researchers, and the
increasing reliance on biological processes and plant interactions
with microbes through ‘ecological intensification’ may be one of the
most promising strategies to overcome these problems (Yang et al.
2014). Bio-fertilizers containing efficient microorganisms, improve
plant growth in many ways compared to synthetic fertilizers, by way
of enhancing crop growth, sustainability of environment and crop
productivity and efficient microbes with PGPR and endophytic
bacteria have been produced as bio-fertilizer for crop production in
sustainable agriculture (Bhardwaj et al. 2014).
In the Mekong Delta is the main area of food production of
crucial importance of ensuring national food security and
agricultural product export (Dan et al., 2015), it has more than 4
millions hectare but area of rice production only occupied 1,9128
millions hectare (46.9%)(MRE, 2014) and acid sulphate soil
occupied 4 millions hectare with 4 regions

Chapter 2: Materials and methods
2.1 Materials
Rice, sweet potato samples were collected from Long Xuyen
Quadrangle, Plain of Reed, Depressed of Hau River and Ca Mau
Peninsula, Mekong Delta, Vietnam, their soils were also collected at
corresponding sites. Media were used to isolate plant associated
bacteria at rice, sweet potato and rhizospheric soil as Burk, NBRIP,
LGI, NFb. Bacterial identification from rhizospheric soil: use a pair
of primers as Forward Primer 8F and Reverse Primer 1492R (Turner
et al., 1999)
5


8F: 5’- AGAGTTTGATCC TGGCTCAG-3’.
1492R:5’-TACGGTTACCTTGTTACGACTT-3’.
Bacterial identification from rice, sweet potatoendophytic
bacteria: use a pair of primers as P515FPL and P13B (Zinniel et al.,
2002):
P515FPL:

5’- GTGCCAGCAGCCGCGGTA A -3’.

P13B:

5’- AGGCCCGGG AACGTATTCAC -3’.

2.2 Methods
2.2.1 Sample collection
Rice and rice rhizospheric soil samples were collected from
Long Xuyen Quadrangle, Plain of Reed, Depressed of Hau River and

Ca Mau Peninsula. For sweet potato samples were also collected
from 4 areas, the exception for Ca Mau Peninsula.
2.2.2 Isolation and selection of endophytes with nitrogen fixation
and P solubilization
Media were used to isolate plant associated bacteria including
Burk, NBRIP, LGI, NFb; for biofertility activities were Burk’N free;
and insoluble P as Al-P and Fe-P were added to NBRIP’P free.
2.2.3 Bacterial identification
The selected bacteria isolated from both plant and soil were
identified by 16S rDNA sequence analysis. The amplified PCR
products were purified using a Invitrogen Kit as described by the
manufacturer’s guide. The sequencing results along with the
chromatograms were analyzed using BioEdit, version 7.0.5.3 and
ChromasPro version 1.7. The corrected sequences were compared
tothe available sequences in the GenBank database for the
determination of the most similarity by Basic Local Alignment
Search Tool (BLAST) of National Center for Biotechnology
Information (NCBI) website. Multiple sequence alignments were
done using CLUSTALW then neighbor-joining phylogenetic tree
was reconstructed using MEGA software, version 6.06, wherein
6


evolutionary distance matrix was calculated by Jukes–Cantor model
and topologies of the neighbor-joiningtrees were calculated by
bootstrap resampling method based on 1,000 replicates.
2.2.4 Selection of potential bacteria for field application
The indentified bacteria sequenced was used to confirm the ability
for nitrogen fixation from Burk’s N free and phosphorus
solubilization from Al-P and Fe-P instead of adding Ca-P under

acidic medium; 3 strains for each plant was selected to apply on acid
sulfate soil in paddy field and sweet potato.
2.2.5 Evaluation of efficacy of selected bacterial strains on paddy
field on acid sulfate soils
Experiment 1: Effects of selected bacterial strains (VK1, VK2, VK3)
and nitrogen fertilizer rates on rice yield in summer-autumn 2015
A 3 x 3 factorial experiment was carried out in a completely
randomized block design including the main factor of inoculants
(VK1, VK2, VK3) and the minor factor as N fertilizer rates (30N,
60N, 90N) to have a total of 9 treatments, with 4 replications from
20 m2 for each plot. Treatments are shown in Table 2.1
Table 2.1: Effects of selected bacterial strains and nitrogen fertilizer rates
on rice yield in summer-autumn 2015
N rate (kg/ha)
30
60
90

Tested bacterial strains
VK1
NT1
NT4
NT7

VK2
NT2
NT5
NT8

VK3

NT3
NT6
NT9

P and K fertilizes was applied as recommendation as follows: 60P2O5
- 30K2O kg ha-1
Experiment 2: Evaluation of promised bacterial strains on rice yield
in autumn–winter 2015 in the Mekong Delta
An experiment has been conducted in a completely randomized
block design including six treatments, with 4 replications from 20 m 2
for each plot. Treatments are shown in Table 2.2
7


Table 2.2: Evaluation of promised bacterial strains and N fertilizer rates
rice yield in autumn –winter 2015
No
Description
Treatment
1
00-60-30
No added nitrogen
2
90-00-30
No added phosphorus
3
90-60-30
NPK
4
30-60-30 + VKX

30 kg N/ha + VKX
5
60-60-30 + VKX
60 kg N/ha+ VKX
6
90-60-30 + VKX
90 kg N/ha+ VKX
* VKX was selected from experiment 1 (summer-autumn 2015)

Experiment 3: Effects of selected bacterial strains and phosphorus
fertilizer rates on rice yield in autumn –winter 2015
A 3 x 3 factorial experiment was carried out in a completely
randomized block design including the main factor of inoculants
(VK1, VK2, VK3) and the minor factor as P fertilizer rates (30 P2O5,
60 P2O5, 90 P2O5) to have a total of 9 treatments, with 4 replications
from 20 m2 for each plot. Treatments are shown in Table 2.3
Table 2.3: Effects of selected bacterial strains and phosphorus fertilizer
rates on rice yield in summer-autumn 2015
Tested bacterial strains
P2O5rate (kg/ha)
VK1
VK2
VK3
30
NT1
NT2
NT3
60
NT4
NT5

NT6
90
NT7
NT8
NT9

2.6 Evaluation of efficacy of selected bacterial strains on sweetpotato field on acid sulfate soils
Experiment 1: Effects of selected bacterial strains (VK4, VK5, VK6)
and nitrogen fertilizer rates on sweet-potato yield in spring-summer
2016
A 3 x 3 factorial experiment was carried out in a completely
randomized block design including the main factor of inoculants
(VK4, VK5, VK6) and the minor factor as N fertilizer rates (30N,
60N, 90N) to have a total of 9 treatments, with 4 replications from 5
m2 (5m x 1m) for each plot. Treatments are shown in Table 2.4
8


Table 2.4: Effects of selected bacterial strains combined nitrogen fertilizer
rates on sweet-potato yield in spring-summer 2016
Tested bacterial strains
N rate (kg/ha)
VK4
VK5
VK6
30
NT1
NT2
NT3
60

NT4
NT5
NT6
90
NT7
NT8
NT9

P and K fertilizes was applied as recommendation as follows: 60P2O5
- 30K2O kg ha-1
Experiment 2: Evaluation of promised bacterial strains on sweetpotato yield in autumn –winter 2016
An experiment has been conducted in a completely randomized
block design including six treatments, with 4 replications from 5m2
for each plot. Treatments are shown in Table 2.5
Table 2.5: Evaluation of promised bacterial strains on sweet-potato yield in
autumn –winter 2016
Description
Treatment
00-90-90
No added nitrogen
90-00-90
No added phosphorus
90-90-90
NPK
30-90-90+VKX
30 kg N/ha + VKX
60-90-90+VKX
60 kg N/ha + VKX
90-90-90+VKX
90 kg N/ha + VKX

*VKX was selected from experiment 1 (spring-summer 2016)

Experiment 3: Effects of selected bacterial strains (VK4, VK5,
VK6)and phosphorus fertilizer rates on sweet-potato yield in
summer-autumn 2016
A 3 x 3 factorial experiment was carried out in a completely
randomized block design including the main factor of inoculants
(VK4, VK5, VK6) and the minor factor as P fertilizer rates (30 P2O5,
60 P2O5, 90 P2O5) to have a total of 9 treatments, with 4 replications
from 5 m2 (5m x 1m) for each plot. Treatments are shown in Table
2.6
9


Table 2.6: Effects of selected bacterial strains and phosphorus fertilizer
rates on sweet-potato yield in summer-autumn 2016
Tested bacterial strains
P2O5 rate (kg /ha)
VK4
VK5
VK6
30
NT1
NT2
NT3
60
NT4
NT5
NT6
90

NT7
NT8
NT9

N and K fertilizes was applied as recommendation as follows: 100N30K2O kg ha-1.
2.3 Statistical analysis
The data shown in this paper are mean values of four
replications, unless otherwise stated. The data were subjected to twoway analysis of variance (ANOVA) using SPSS software version
13.0. Means were separated by ANOVA and the significant
differences were assessed by Duncan’s post-hoc test at P< 0.05.
Chapter 3: Results and discussion
3.1 Isolation and selection of endophytic bacteria and
rhizobacterial strains for nitrogen fixing and phosphorus
solubilizing from acid sulfate soil
A total of 431 rice endophytic bacterial strains including 272
strains isolated from rhizobacterial strains and 159 endophytic
bacterial strains were obtained. Morphological properties of colonies
had colorless, opaque, yellow and very yellow. Besides, colony
morphology was mainly circular shapes and entire margin, rod shape
and Gram-negative. A total of 424 sweet potato endophytic bacterial
strains consisting of 271 strains isolated from rhizobacterial strains
and 153 endophytic bacterial strains were obtained. Morphological
characteristics of colonies also were transparency, milk-color, yellow
and very yellow. Moreover, colony morphology was mostly circular
shapes and entire margin, rod shape and Gram-positive, but several
strains had sphere shape. All bacterial strain possesses the ability of
nitrogen fixing and phosphorus solubilizing. The 25 selected rice
endophytic bacterial strains had promised high abilities for nitrogen
fixing, phosphate-solubilizing that were identified by 16S rDNA
10

sequence analysis. The phylogenetic analysis is belonged to Bacilli,
Gammaproteobacteria and Betaproteobacteria groups. Similarly, The
12 selected sweet potato, yam, and cassava endophytic bacterial
strains had high promised capacities for nitrogen fixing, phosphatesolubilizing, which were identified by 16S rDNA sequence analysis.
The phylogenetic analysis is closely classified to Bacilli and
Gammaproteobacteria groups.
From selected 15 and 20 rice endophytic bacteria and
rhizobacterial strains having high nitrogen fixation capacity and P
solubilization, respectively, under neuter broth (pH 7.0), they were
used to evaluate both abilities under acidic medium (pH 4.0). The
results showed that all strains have ability of nitrogen fixation, and P
solubilization from Al-P and Fe-P.
Strains KG2-21, HG6-21b and BL1-21b produced 0.79 –
1.38 mg NH4+ L-1 at 8 DAI. Similarly, PO43- was produced 22.2 –
58.1 from Al-P source and 24.9 – 48.9 mg L -1 from Fe-P source
(Table 3.1).
Table 3.1: Ability of nitrogen fixation and P solubilization from Al-P and
Fe-P sources at pH 4.0 by selected endophytic rhizobacterial strains in rice
Days after incubation
ASS area
Strain
NH4+ (mg L-1)
2
4
6
8
KG2-21
1.51

1.49
1.83
1.37
AG5-13
0.25
0.66
0.88
0.73
Long
DT10-12a
0.33
0.37
0.42
0.34
Xuyen
AG8-13
0.28
0.30
0.35
0.29
Quadrang AG9-22
0.16
0.21
0.22
0.25
le
KG5-3a
0.20
0.23
0.21

0.25
AG5-3
0.21
0.23
0.22
0.25
AG9-4b
0.17
0.23
0.18
0.27
Plain of LA2-21b
0.45
0.52
0.60
0.56
Reed
LA5-22b
0.17
0.39
0.34
0.33
DT9-11
0.26
0.35
0.33
0.43
LA4-3b
0.31
0.29

0.36
0.29
LA5-3
0.25
0.40
0.41
0.34

11


Depresse
d of Hau
River

Ca Mau
Peninsula

ASS area

Long
Xuyen
Quadrang
le

Plain of
Reed

Depresse
d of Hau

River

LA4-3c
HG6-21b
HG9-3a
HG10-3a
VL3-21
VL4-12
VL3-3b
VL3-3c
BL1-21b
BL5-12
CM7-22
BL2-21b
BL3-4a
BL4-3
BL5-4b
BL5-3

0.22
1.06
0.47
0.32
0.15
0.12
0.19
0.16
1.51
0.44
0.11

0.18
0.17
0.17
0.33
0.27

Strain
KG2-21
AG5-13
DT10-12a
AG8-13
AG9-22
KG5-3a
AG5-3
AG9-4b
LA2-21b
LA5-22b
DT9-11
LA4-3b
LA5-3
LA4-3c
HG6-21b
HG9-3a
HG10-3a
VL3-21
VL4-12
VL3-3b

5
73.9

10.03
21.35
12.98
14.56
11.26
11.70
10.32
24.5
14.21
9.58
11.05
18.06
11.00
145.0
44.3
33.2
15.1
7.9
11.8

12

0.30
0.28
0.91
0.81
0.64
0.60
0.28
0.54

0.33
0.62
0.29
0.44
0.18
0.42
0.15
0.35
1.48
1.82
0.76
0.78
0.33
0.28
0.16
0.33
0.32
0.20
0.13
0.18
0.31
0.31
0.24
0.47
P-Fe (mg L-1)
10
15
32.8
27.5
9.00

8.70
18.68
16.36
4.33
3.85
5.47
4.61
10.50
10.54
10.90
10.90
14.21
15.55
10.1
9.2
7.00
6.37
3.72
3.35
4.67
5.80
10.84
11.88
9.28
9.11
63.2
55.8
26.6
29.1
22.5

25.6
16.8
22.1
9.8
17.8
8.9
4.5

0.31
0.79
0.81
0.45
0.33
0.37
0.38
0.32
1.38
0.62
0.51
0.49
0.21
0.21
0.33
0.34
20
22.2
9.95
13.36
2.60
3.33

13.60
13.30
16.69
5.4
7.15
1.78
3.78
14.04
4.12
58.1
34.4
22.6
22.3
18.8
1.0


Ca Mau
Peninsula

ASS area

Long
Xuyen
Quadrang
le

Plain of
Reed

Depresse
d of Hau
River
Ca Mau
Peninsula

VL3-3c
BL1-21b
BL5-12
CM7-22
BL2-21b
BL3-4a
BL4-3
BL5-4b
BL5-3

9.8
135.7
22.8
16.9
13.4
10.14
10.50
1.78
2.28

Strain
KG2-21
AG5-13
DT10-12a

AG8-13
AG9-22
KG5-3a
AG5-3
AG9-4b
LA2-21b
LA5-22b
DT9-11
LA4-3b
LA5-3
LA4-3c
HG6-21b
HG9-3a
HG10-3a
VL3-21
VL4-12
VL3-3b
VL3-3c
BL1-21b
BL5-12
CM7-22
BL2-21b
BL3-4a
BL4-3
BL5-4b

5
7.5
3.1
2.20

2.10
1.30
0.88
0.80
0.72
2.7
3.8
2.6
5.2
5.5
5.1
18.4
2.7
1.6
1.2
0.6
1.4
1.2
19.6
3.4
1.2
1.0
0.88
1.80
1.12

13

7.4
3.8

48.1
54.2
13.2
11.0
12.1
9.8
15.8
5.8
14.62
12.10
13.80
10.25
0.77
0.95
1.14
1.04
P-Al (mg L-1)
10
15
9.3
11.1
3.8
4.0
4.10
2.80
4.00
3.00
2.30
3.20
1.37

1.40
1.47
1.32
1.46
1.28
4.7
5.1
2.2
1.9
2.3
2.0
2.7
2.4
3.0
2.9
2.8
2.4
18.3
16.2
4.7
5.1
1.9
3.9
1.1
1.2
0.8
1.4
0.9
1.8
0.8

1.5
18.8
21.3
5.6
6.7
0.6
3.2
0.5
1.9
1.48
1.35
1.51
1.38
0.49
0.41

0.8
54.4
6.6
4.4
1.2
16.38
17.00
0.63
1.16
20
24.9
4.7
8.90
8.00

6.20
1.21
1.25
1.27
12.4
1.2
1.6
2.8
3.4
3.1
45.8
12.4
7.8
6.3
1.7
5.2
4.3
48.9
11.5
4.3
5.6
1.24
1.37
0.33


BL5-3

0.36

0.22

0.19

0.12

Selection of bacterial nitrogen fixer and phosphorus
solubilizer for sweet potato.
The results of selection bacterial nitrogen fixer and
phosphorus solubilizer from Al-P and Fe-P source for sweet potato
from 8 nitrogen fixer strains and 12 phosphorus solubilizer strains of
endophytic and rhizopheric bacteria. See in details, strains KG2-32,
TG7-2-22 and HG7-2-12 produced 0.18 - 2.53, 9.94 - 13.73 and 7.59
- 11.88 mg L-1, respectively (Table 3.2).
Table 3.2: Ability of nitrogen fixation and P solubilization from Al-P and
Fe-P sources at pH 4.0 by selected endophytic rhizobacterial strains in
sweet potato

ASS area
Long Xuyen
Quadrangle

Plain of Reed

Depressed of Ha
u River
ASS area
Long Xuyen
Quadrangle
Plain of Reed

Days after incubation

Strain
KG2-32
KG9-211
AG1-111
AG9-31
TG7-2-22
LA2-33
LA1-33
LA2-113
HG7-2-12
VL9-42
VL5-211
HG5-32
Strain
KG2-32
KG9-211
AG1-111
AG9-31
TG7-2-22
LA2-33
LA1-33

2
0.84
0.34
0.41
0.21

0.72
0.28
0.18
0.28
0.62
0.40
0.32
0.34
5
9.47
4.24
3.64
3.62
9.01
4.42
1.71

14

NH4+ (mg L-1)
4
6
1.09
2.15
0.23
0.39
0.30
0.34
0.24
0.28

0.82
1.73
0.31
0.48
0.20
0.23
0.26
0.26
0.77
1.49
0.52
0.26
0.29
0.29
0.38
0.36
P-Fe (mg L-1)
10
15
9.85
11.48
2.55
8.41
2.16
2.03
2.11
1.77
9.83
7.58
4.88

5.70
1.02
0.87

8
2.28
0.44
0.33
0.25
2.53
0.61
0.22
0.28
2.18
0.40
0.32
0.31
20
11.60
6.21
2.51
1.43
13.73
6.08
0.55


Depressed of Ha
u River
ASS area

Long Xuyen
Quadrangle

Plain of Reed

Depressed of Ha
u River

LA2-113
HG7-2-12
VL9-42
VL5-211
HG5-32
KG2-32
KG9-211
AG1-111
AG9-31
TG7-2-22
LA2-33
LA1-33
LA2-113
HG7-2-12
VL9-42
VL5-211

5
3.18
2.36
1.08
0.83

4.63
2.96
0.53
0.26
5.52
1.26
0.25

1.05
0.92
8.75
4.03
3.80
9.52
1.51
1.27
1.34
1.27
P-Al (mg L-1)
10
15
5.29
7.75
2.81
3.07
0.56
0.40
0.48
0.45
4.75

9.68
3.07
3.58
0.46
0.44
0.33
0.34
6.09
7.11
1.84
2.73
0.21
0.19

HG5-32

1.04

0.43

Strain

1.20
5.91
3.71
3.37
1.55

0.34

0.75
9.94
7.76
1.03
1.04
20
10.50
4.28
0.58
0.57
11.88
3.62
0.36
0.42
7.59
3.10
0.15

0.15

To obtain the maximum efficacy, one bacterium was selected
for each area due to adaptive property of indigenous soil. Thus,
strains KG2-21, HG6-21band BL1-21b were selected for Long
Xuyen Quadrangle, Depressed of Hau River and Plain of Reed,
respectively. They are the best strain for each area, with P
concentration as 47.8, 98.5 and 88.8 mg L-1, respectively. Moreover,
all selected strains have ability of nitrogen fixation. Particularly,
strains HG6-21band BL1-21b also produced the highest NH4+ for
each area. Strains KG2-21, HG6-21b and BL1-21b were identified as
Burkholderia vietnamiensis X1, Burkholderia vietnamiensis X2 and

Burkholderia vietnamiensis X3, respectively.
Similarly, strains KG2-32, TG7-2-22, and HG7-2-12 were
selected for sweet potato in Long Xuyen Quadrangle, Plain of Reed
and Depressed of Hau River, respectively. They were identified as
Enterobacter cloacae X4, Burkholderia acidipaludis X5 and
Bacillus sp. X6. Strains KG2-32 and HG7-2-12 were selected
because they are the best strain to fix nitrogen and also possess the
15


ability of P-solubilization in 15 highest strains. For TG7-2-22, it
belongs to acid-resistant bacterium that is a special property of ASS.
3.2 Evaluation the effects of bacterial N-fixer and P solubilizer
on rice and sweet potato yield
3.2.1 Effects of selected bacterial strains B. vietnamiensis X1, X2,
X3 and nitrogen fertilizer rates on rice yield in ASS in summerautumn 2015
The research results showed that the number of panicles per
m and the number of filled grains per panicle were found to increase
under the treatment of Burkholderia vietnamiensis X3 in 2015 SA
season at Hong Dan and Long My districts. As a result, the rice yield
obtained from the Burkholderia vietnamiensis X3 bacterium
treatment was highest among the three treated bacteria. In Hon Dat
district, however, the most efficiency was found in the soil treated
with Burkholderia vietnamiensis X1 bacterium. In 2015 AW season,
the treatment of Burkholderia vietnamiensis X3 bacterium in
combination with 60 kgN ha-1 brought about the rice yield higher
than the treatment of 90 kgN ha-1 alone. The research results
indicated that application of phosphorus fertilizer alone to acid
sulfate soils did not affect rice yields. However, a combined
application of phosphorus fertilizer and Burkholderia vietnamiensis

X1 and Burkholderia vietnamiensis X3 bacteria resulted in the
highest yield in Hon Dat district and Hon Dan district, respectively.
2

Table 3.3: Effects of selected bacterial strains and nitrogen fertilizer rates
on rice yield components and yield in summer-autumn 2015 in acid sulfate
soil
Site

Long
My –
Hau
Gian
g

Factor

Nitrogen
rates (A)

Treatmen
t

30 N
60 N
90 N

Grai
n
yield

(ton
ha-1)

5.45
b

6.41
a

6.52

Numbe
r of
panicle
m-2

Total
spikelet
s
panicle-

1.000
grain
(g)

1

Filled
spikelet
percentag

e (%)

460b

89b

64.4b

23.6

510a

101a

70.6a

23.7

a

a

b

23.7

528

a

16

105

63.0


VK1
Tested
bacteria(B
)

VK2
VK3

F (A)
F (B)
F (A*B)
CV (%)
30 N
Nitrogen
rates (A)

60 N
90 N

Hon
Dat –
Kien
Gian

g

VK1
Tested
bacteria
(B)

VK2
VK3

F (A)
F (B)
F (A*B)
CV (%)
30 N
Nitrogen
rates (A)

60 N
90 N

Hong
Dan
– Bac
Lieu

VK1
Tested
bacteria
(B)

VK2
VK3

F (A)
F (B)
F (A*B)
CV (%)

6.02

481b

100

68.2

23.7

481b

96

66.9

23.8

537a

100

62.9

23.7

**
**
*
5.48

*
ns
ns
11.6

*
ns
ns
8.86

ns
ns
ns
7.24

372b

80.9b

79.1

25.8

473a

94.5a

84.4

25.7

478a

96.3a

76.4

25.2

476a

102a

81.1

25.8

422b

86.1b

82.1

25.7

426b

82.8b

76.7

25.3

**
*
ns
9.66

**
**
ns
10.8

ns
ns
ns
8.74

ns
ns

ns
7.64

424b

97b

68.6

24.6

481a

106ab

70.9

23.3

494a

114a

71.1

24.1

460b

97b

69.2

23.6

444b

101b

72.1

23.9

a

494a

119a

69.3

24.3

**
**
*
6.16

**
**

**
5.34

**
**
ns
9.32

ns
ns
ns
13.0

ns
ns
ns
6.04

b

5.89
b

6.53
a

**
*
*
6.99

3.95
b

4.76
a

4.81
a

4.89
a

4.21
b

4.41
b

**
*
ns
10.1
5.20
b

5.77
a

5.63
a

5.37
b

5.36
b

5.86

17


Values are means of four replications. Different lowercase letters in the same
column indicate significant differences at P<0.01 (**), <0.05 (*); and ns is no
significant difference at P>0.05.
VK1: Burkholderia vietnamiensis X1; VK2: Burkholderia vietnamiensis X2;
VK3: Burkholderia vietnamiensis X3

Table 3.4: Effects of selected bacterial strains and phosphorus fertilizer
rates on rice yield components and yield in autumn –winter 2015 in acid
sulfate soil
Site

Factor

P rates
(A
Long
My –
Hau

Gian
g

Tested
bacteri
a (B)
F (A)
F (B)
F (A*B)
CV (%)
P rates
(A

Hon
Dat –
Kien
Gian
g

Hong
Dan –
Bac
Lieu

Tested
bacteri
a (B)
F (A)
F (B)
F (A*B)

CV (%)
P rates
(A
Tested
bacteri
a (B)
F (A)
F (B)

Treatment

30 P2O5
60 P2O5
90 P2O5
VK1
VK2
VK3

30 P2O5
60 P2O5
90 P2O5
VK1
VK2
VK3

30 P2O5
60 P2O5
90 P2O5
VK1
VK2

VK3

Grain
yield
(ton
ha-1)

Numbe
r of
panicle
m-2

Total
spikelet
s
panicle-1

Filled
spikelet
percentag
e (%)

1.000
grain
(g)

6.37
6.37
6.65
6.17b

6.52a
6.70a
ns
*
*
5.22
4.98
4.60
4.83
5.40a
4.10b
4.90b
ns
**
ns
9.72
5.66
5.43
5.59
5.42b
5.10b
5.96a
ns
**

538
549
558
548
536

562
ns
ns
ns
4.80
503
481
497
568a
461b
452b
ns
**
ns
13.7
511
496
523
507
515
509
ns
ns

107
100
108
110
99
106

ns
ns
ns
11.7
97.8
92.1
88.5
108a
78.5b
91.1b
ns
**
ns
16.2
98
105
103
101
99
107
ns
ns

62.1
62.9
65.8
59.1
66.2
63.2
ns

ns
ns
11.3
82.1
84.4
82.0
84.9a
79.4b
84.2a
ns
*
ns
4.63
63.6
63.4
66.5
62.8b
63.2b
67.7a
ns
*

23.7
23.9
24.1
23.7
24.1
23.9
ns
ns

ns
5.21
25.8
25.7
24.6
24.7
25.1
26.3
ns
ns
ns
8.87
24.3
23.3
24.1
24.1
23.7
23.9
ns
ns

18


F (A*B)
ns
ns
ns
ns
ns

CV (%)
6.65
10.9
10.3
5.91
4.51
Values are means of four replications. Different lowercase letters in the same
column indicate significant differences at P<0.01 (**), <0.05 (*); and ns is no
significant difference at P>0.05.
VK1: Burkholderia vietnamiensis X1; VK2: Burkholderia vietnamiensis X2;
VK3: Burkholderia vietnamiensis X3

Table 3.5: Effects of promised bacterial strains B. vietnamiensis X1andB.
vietnamiensisX3 on rice yield components and yield in summer-autumn
2015 in acid sulfate soil
Site

Long
My –
Hau
Giang

Hon
Dat –
Kien
Giang

Hong
Dan –
Bac

Lieu

Factor

00-60-30
90-00-30
90-60-30
30-6030+VK3
60-6030+VK3
90-6030+VK3
F
CV (%)
00-60-30
90-00-30
90-60-30
30-6030+VK1
60-6030+VK1
90-6030+VK1
F
CV (%)
00-60-30
90-00-30
90-60-30
30-60-

Treatme
nt

Grain
yield

(ton ha-1)

Numb
er of
panicl
e m-2

Total
spikelets
panicle-1

Filled
spikelet
percentag
e (%)

4.68c
5.67ab
6.07a

374c
525ab
528ab

69b
105a
104a

56.1
64.8

61.2

23.4
23.9
24.4

5.25bc

484b

89b

65.1

23.9

6.18a

549a

107a

74.1

23.6

6.33a

529ab

111a

64.9

23.6

**
7.29
3.14c
4.31b
4.35b

**
6.05
355b
454a
503a

**
8.01
62.7c
85.3b
110a

ns
10.4
79.9
77.5
73.3

ns
5.56
25.3
24.5
25.3

4.52b

505a

85.6b

83.1

26.2

5.02a

522a

108a

83.5

25.0

4.60b

502a

109a

81.6

23.7

**
5.31
3.43c
5.53ab
5.47ab
5.07b

**
8.32
349c
513a
511a
433b

**
9.47
82c
92bc
109ab
110ab

ns
5.95
69.7

63.6
70.6
69.1

ns
5.28
24.1
24.1
25.1
25.3

19


30+VK3
60-6030+VK3
90-6030+VK3
F
CV (%)

5.77a

523a

121a

66.3

23.4

5.86a

527a

113ab

72.7

24.3

**
5.54

**
5.74

*
11.4

ns
5.97

ns
5.76

Values are means of four replications. Different lowercase letters in the same
column indicate significant differences at P<0.01 (**), <0.05 (*); and ns is no
significant difference at P>0.05.
VK1: Burkholderia vietnamiensis X1; VK3: Burkholderia vietnamiensis X3

3.2.2 Efficacy of promising bacterial strains with nitrogen
fixation and phosphorus solubilization as E. cloacae X4, B.
acidipaludis X5, Bacillus sp. X6 on sweet-potato yield on acid
sulfate soils in the field
In this study, the influence of plant-associated bacteria (Enterobacter
cloacae, Burkholderia acidipaludis, Bacillus sp.) combined with the
nitrogen, phosphate fertilizers dose on sweet potato yield and
efficiency of promising plant-associated bacteria to sweet potatoes
yield cultivated on acid sulfate soils in the Mekong Delta was
investigated. The field experiment was conducted over a two crops:
spring-summer 2016 and summer- autumn 2016. The results showed
that the bacteria Burkholderia acidipaludis have high nitrogen
fixation capability compared to those in the 2 other bacteria through
increased number of tubers and sweet potato yield. Applying
60kgN/ha and adding Burkholderia acidipaludis showed that the
tuber number, tuber length, tuber diameter and sweet potato yield
was equivalent with applying 90 kg N/ha. Use of Burkholderia
acidipaludis can save 30% of nitrogen fertilizers for sweet potato.
Table 3.6: Effects of selected bacterial strains and nitrogen fertilizer rates
on tuber number, tuber length, tuber diameter and sweet potato yield in
spring- summer 2016
Site

Factor

Treatme
nt

Sweet potato
yield

20

Tuber
number

Tuber
length

Tuber
diameter


(tons ha-1)
(5 m2)
(cm)
(cm)
b
30 N
13.4
42b
10.2b
4.02b
N rates
60 N
17.6a
68a
12.1a
4.90a
(A)

a
a
a
90 N
17.7
68
11.9
4.70a
VK4
15.6b
61
10.7b
4.35b
Tested
bacteri
VK5
17.5a
61
12.7a
4.81a
Tri Ton, An
b
b
a (B)
Giang
VK6
15.6
56
10.6
4.46b

F (A)
**
**
**
**
F (B)
*
ns
**
**
F (A*B)
*
**
**
**
CV (%)
9,85
7.89
7.41
5.86
30 N
12.7b
49b
12.3b
4.17b
N rates
a
a
a
60 N

18.9
77
15.3
4.91a
(A)
a
a
a
90 N
19.2
74
15.1
5.02a
VK4
16.5b
65b
12.9b
4.78a
Tested
a
a
a
bacteri
VK5
18.1
72
15.1
5.04a
Tan Phuong
b

b
a
a (B)
– Tien Giang
VK6
16.1
63
14.8
4.28b
F (A)
**
**
**
**
F (B)
**
**
**
**
F (A*B)
*
*
*
*
CV (%)
5,20
7.74
6.85
6.28
30 N

15.4b
70b
11.3b
4,33b
N rates
a
a
a
60 N
20.4
94
12.5
4,85a
(A)
a
a
a
90 N
20.8
96
12.4
4,98a
b
b
b
b
VK4
17.2
81
11.7

4,51
Tested
bacteri
VK5
21.3a
96a
12.7a
5,24a
Binh Tan –
a (B)
Vinh Long
VK6
18.2b
83b
11.7b
4,98b
F (A)
**
**
**
**
F (B)
**
*
*
**
F (A*B)
*
*
ns

ns
CV (%)
7,91
11.34
6.54
7.36
Values are means of four replications. Different lowercase letters in the same column indicate
significant differences at P<0.01 (**), <0.05 (*); and ns is no significant difference at
P>0.05.
VK4:Enterobacter cloacae X4; VK5:Burkholderiaacidipaludis X5; VK6: Bacillus sp. X6.

Table 3.7: Effects of selected bacterial strains and phosphorus fertilizer
rates on tuber number, tuber length, tuber diameter and sweet potato yield
in summer –autumn 2016
Site

Factor

Treatment

Sweet potato
yield
(tons ha-1)

21

Tuber
number
(5 m2)

Tuber
length
(cm)

Tuber
diameter
(cm)


30 P2O5
13.2b
44b
10.2b
4.38b
P
a
a
a
rates
60 P2O5
18.2
66
12.5
4.75a
a
a
a
(A)
90 P2O5
18.4

68
12.8
5.25a
16.1b
55b
11.6
4.54b
Tested VK4
17.6a
66a
12.4
5.25a
Tri Ton, An bacter VK5
b
b
ia (B) VK6
Giang
16.2
58
11.6
4.58b
F (A)
*
**
**
**
F (B)
*
*
ns

*
F (A*B)
*
*
ns
*
CV (%)
7,10
14.18
8.84
10.53
30 P2O5
16.1b
48b
12.9b
4.01b
P
a
a
a
rates
60 P2O5
18.4
71
15.3
4.82a
a
a
a
(A)

90 P2O5
18.7
76
14.8
4.95a
17.2b
61b
14.0b
4.70a
Tested VK4
Tan Phuoc
a
a
a
bacter VK5
18.9
71
15.6
4.69a
– Tien
b
b
b
ia (B) VK6
17.1
63
13.4
4.39b
Giang
F (A)

**
**
**
**
F (B)
*
**
**
*
F (A*B)
*
ns
ns
*
CV (%)
7,62
9.06
7.53
5.15
30 P2O5
16.4b
82b
10.5b
4.30b
P
a
a
a
rates
60 P2O5

19.7
96
11.8
4.96a
a
a
a
(A)
90 P2O5
19.5
96
11.7
4.86a
b
b
b
VK4
18.1
86
11.3
4,60
Tested
19.7a
98a
11.7
5,03a
Binh Tan – bacter VK5
ia (B) VK6
Vinh Long
18.1b

90b
10.9
4,50a
F (A)
**
**
**
**
F (B)
*
*
ns
**
F (A*B)
*
*
ns
**
CV (%)
7,66
8.12
6.78
5.85
Values are means of four replications. Different lowercase letters in the same column
indicate significant differences at P<0.01 (**), <0.05 (*); and ns is no significant
difference at P>0.05.
VK4:Enterobacter cloacae; VK5:Burkholderiaacidipaludis; VK6: Bacillus sp.

Table 3.8: Evaluation of promised bacterial strains on tuber number, tuber
length, tuber diameter and sweet potato yield in summer –autumn 2016

Site
Tri Ton,
An Giang

Treatment
00-90-90
90-00-90
90-90-90
30-90-90+X5
60-90-90+X5
90-90-90+X5

Tuber
number
(5 m2)
40d
54b
65a
47c
68a
68a

22

Tuber
length
(cm)
9.3c
10.3bc
12.1ab

11.9ab
13.4a
12.8a

Tuber
diameter
(cm)
3.63d
4.33bc
4.98ab
4.27c
5.01ab
5.20a

Sweet potato
yield
(tons ha-1)
10.8c
15.7b
18.3a
15.5b
19.1a
18.3a