MINISTRY OF EDUCATION AND TRAININGCAN THO UNIVERSITY DOCTORAL THESIS SUMMARY Specialized: Soil Science Code: 9620103 LY NGOC THANH XUAN ISOLATION, SELECTION AND IDENTIFICATION PLANT ASSO
Trang 1MINISTRY 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
Trang 2THE 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 atbasic level
Meeting at: Meeting room 3, 2nd floor, administrative house, CanTho 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
Trang 3LIST 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 riceendophytic 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 rhizosphericbacteria 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 endophyticand rhizopheric bacteria associated sweet-potato plants
cultivated on soils of the Mekong delta, Vietnam World journal of Pharmacy and Pharmaceutical sciences, 6 (1): 129- 149.
5 Ly Ngoc Thanh Xuan, Le Van Dang, Tran Van Dung and Ngo
Ngoc Hung, 2018.Applcation of plantassociated bacteria onthe 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.
Trang 41 Rationale and background
Nitrogen (N) is the most essential nutrient to promote rapid plantgrowth and grain yield; thereby, high amounts of chemical Nfertilizers have been applied to gain high crop yield in agriculturalecosystems for intensive fertilization practices (Galloway et al.,2004), especially in low-fertility soils like ASS as an example Thisissue faces not only environmental problems as greenhouse gasemission but also microbial communities as changes of N2-fixergrowth and activity (Reardon et al., 2014; Tang et al., 2017).Therefore, a biological nitrogen source should be altered to tacklewith this problem and make a use of natural resources Specifically,biological nitrogen fixation (BNF) is one of the important naturalprocesses to fix N2 from the atmosphere into bioavailable NH4+ in thesoil 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)forrice has been observed in ASS due to immobilized P asitsprecipitation with free aluminum and iron ions to form aluminumphosphate (AlPO4) and iron phosphate (FePO4) (Margenot et al.2017); and this leads toless available forms of P for plants (Rengeland Marschner 2005) and altered the soil microbial communitystructure (Ragot et al 2016; Soman et al 2017) Hence, several tools
to resolve this problem include chemical, physical and biologicalapproaches Rice cultivation in actual ASS faced with toxicity oftoxicity (Högfors-Rönnholm et al 2018) and acidic environment thatcause low yield (Johnston et al 2016) The ASS can be reclaimed byphysical and chemical techniques although there are manyconstraints 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 betackled by the endophytic bacteria and rhizobacterial strains becausethey possesses the ability of N2-fixation, P-solubilization from P-Al,and P-Fe sources Moreover, the efficacy of microorganism isdepended on interaction between bacteria and host (Patnailk, 1994)
Trang 5Regarding above information, to achieve the sustainable agriculturalsystem for solving the serious problems of rice cultivation on ASS,isolation, selection and application of endophytic bacteria andrhizobacterial strains from rice, sweet potato and their soils arenecessary.
2 Objectives
(i) Isolation of endophytic and rhizospheric bacterial strainsassociated with sweet potato and rice for abilities of nitrogen fixing,phosphate-solubilizing;
(ii) Selection of promising nitrogen fixing, solubilizing bacterial strains for improvement of sweet potato andrice growth and yield in the greenhouse and field conditions;
phosphate-(iii) Suggestion of promised 2-3 strains to apply on acidsulfate soil
3 Novelty of research
The without nitrogen fertilizer application decreased riceyield on acid sulfate soil in Long My – Hau Giang, Hon Dat – KienGiang 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 bacteriathrough 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.
Trang 64 Outline of thesis content
The thesis included 103 pages with 5 chapters Chapter 1:Introduction (Pages: 1-5); Chapter 2: Literature review (Pages: 6-35); Chapter 3: Materials and methods (Pages: 36-58); Chapter 4:Results and discussion (Pages: 59-102); Chapter 5: Conclusions andrecommendations (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 onearth (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 produceabout 60% more rice than today’s global production to feed the extrabillion people (Ladha and Reddy, 2003) Increases in the demand forrice, as a result of an increase in population, creates the need toimprove rice productivity and one of the most important factors forhigh yields of rice production are chemicals fertilizers andpesticides, which may cause environmental pollution and negativelyinfluence 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 hugeeconomic and social importance in developing countries (Souza andLorenzi, 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 andnitrogen (N) poor soils (Khan and Doty, 2009) Besides rice and fruitplants, sweet potato plant also is cultivated popularly on alluvial soil
in the dry season as crop for food and exporting in the MekongDelta, Vietnam Sweet potato area occupied 21,500 ha (9.3% sweetpotato area in Vietnam) with yield over 20 tons/ha in the MekongDelta (Agricultural Statastic in 2010)
Trang 7In the kinds of chemical fertilizers, substances composed ofknown quantities of nitrogen, phosphorus and potassium, keep animportant 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 alimiting 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 rootdevelopment, poor flowering and lack of seed etc., consequentlycausing 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 differentmicroprganisms, bacteria are by far the most common (i.e., 95%) Ithas been known for some time that the soil hosts a large number ofbacteria (often around 108 to 109 cells per gram of soil) and that thenumber 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 tomeke 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 inthe formation or modification of soil (Pate and Verboom, 2009;Taylor et al., 2009) and microorganisms present in the rhizosphereplay 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; beneficialmicroorganisms include N2-fixing bacteria in association withlegumes and interaction of roots with mycorrhizal fungi andphosphate-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 bythe following inherent distinctiveness’s: (i) they must be proficient tocolonize the root surface (ii) they must survive, multiply andcompete with other microbiota, at least for the time needed toexpress their plant growth protion/protection activities, and (iii) theymust promote plant growth (Kloepper, 1994)
Trang 8Endophytic bacteria are microorganisms that live in plant tissuesand they may be responsible for the supply of biologically fixednitrogen to their host plant (Boddey et al., 2005) Endophytes alsopromote plant growth by a number of similar mechanisms asphosphate solubilization activity (Waklin et al., 2004), indole aceticacid production (Lee et al., 2004) and the production of asiderophores (Costa and Lopez, 1994) Recently, the potential formicrobes to increases nutrient availability and to enhance cropgrowth has garnered the attention of the researchers, and theincreasing reliance on biological processes and plant interactionswith microbes through ‘ecological intensification’ may be one of themost promising strategies to overcome these problems (Yang et al.2014) Bio-fertilizers containing efficient microorganisms, improveplant growth in many ways compared to synthetic fertilizers, by way
of enhancing crop growth, sustainability of environment and cropproductivity and efficient microbes with PGPR and endophyticbacteria have been produced as bio-fertilizer for crop production insustainable agriculture (Bhardwaj et al 2014)
In the Mekong Delta is the main area of food production ofcrucial importance of ensuring national food security andagricultural product export (Dan et al., 2015), it has more than 4millions hectare but area of rice production only occupied 1,9128millions hectare (46.9%)(MRE, 2014) and acid sulphate soiloccupied 4 millions hectare with 4 regions
Chapter 2: Materials and methods
2.1 Materials
Rice, sweet potato samples were collected from Long XuyenQuadrangle, Plain of Reed, Depressed of Hau River and Ca MauPeninsula, Mekong Delta, Vietnam, their soils were also collected atcorresponding sites Media were used to isolate plant associatedbacteria 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)
Trang 98F: 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.,
2.2.3 Bacterial identification
The selected bacteria isolated from both plant and soil wereidentified by 16S rDNA sequence analysis The amplified PCRproducts were purified using a Invitrogen Kit as described by themanufacturer’s guide The sequencing results along with thechromatograms were analyzed using BioEdit, version 7.0.5.3 andChromasPro version 1.7 The corrected sequences were comparedtothe available sequences in the GenBank database for thedetermination of the most similarity by Basic Local AlignmentSearch Tool (BLAST) of National Center for BiotechnologyInformation (NCBI) website Multiple sequence alignments weredone using CLUSTALW then neighbor-joining phylogenetic treewas reconstructed using MEGA software, version 6.06, wherein
Trang 10evolutionary distance matrix was calculated by Jukes–Cantor modeland topologies of the neighbor-joiningtrees were calculated bybootstrap 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 abilityfor nitrogen fixation from Burk’s N free and phosphorussolubilization from Al-P and Fe-P instead of adding Ca-P underacidic medium; 3 strains for each plant was selected to apply on acidsulfate 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 completelyrandomized 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) Tested bacterial strains
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 randomizedblock design including six treatments, with 4 replications from 20 m2
for each plot Treatments are shown in Table 2.2
Trang 11Table 2.2: Evaluation of promised bacterial strains and N fertilizer rates rice yield in autumn –winter 2015
Experiment 3: Effects of selected bacterial strains and phosphorusfertilizer rates on rice yield in autumn –winter 2015
A 3 x 3 factorial experiment was carried out in a completelyrandomized 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 replicationsfrom 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
P 2 O 5 rate (kg/ha) VK1 Tested bacterial strainsVK2 VK3
A 3 x 3 factorial experiment was carried out in a completelyrandomized 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
Trang 12Table 2.4: Effects of selected bacterial strains combined nitrogen fertilizer rates on sweet-potato yield in spring-summer 2016
N rate (kg/ha) Tested bacterial strains
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
*VK X 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 insummer-autumn 2016
A 3 x 3 factorial experiment was carried out in a completelyrandomized 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 replicationsfrom 5 m2 (5m x 1m) for each plot Treatments are shown in Table2.6
Trang 13Table 2.6: Effects of selected bacterial strains and phosphorus fertilizer rates on sweet-potato yield in summer-autumn 2016
P 2 O 5 rate (kg /ha) Tested bacterial strains
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 272strains isolated from rhizobacterial strains and 159 endophyticbacterial strains were obtained Morphological properties of colonieshad colorless, opaque, yellow and very yellow Besides, colonymorphology was mainly circular shapes and entire margin, rod shapeand Gram-negative A total of 424 sweet potato endophytic bacterialstrains consisting of 271 strains isolated from rhizobacterial strainsand 153 endophytic bacterial strains were obtained Morphologicalcharacteristics of colonies also were transparency, milk-color, yellowand very yellow Moreover, colony morphology was mostly circularshapes and entire margin, rod shape and Gram-positive, but severalstrains had sphere shape All bacterial strain possesses the ability ofnitrogen fixing and phosphorus solubilizing The 25 selected riceendophytic bacterial strains had promised high abilities for nitrogenfixing, phosphate-solubilizing that were identified by 16S rDNA
Trang 14sequence analysis The phylogenetic analysis is belonged to Bacilli,Gammaproteobacteria and Betaproteobacteria groups Similarly, The
12 selected sweet potato, yam, and cassava endophytic bacterialstrains had high promised capacities for nitrogen fixing, phosphate-solubilizing, which were identified by 16S rDNA sequence analysis.The phylogenetic analysis is closely classified to Bacilli andGammaproteobacteria groups
From selected 15 and 20 rice endophytic bacteria andrhizobacterial strains having high nitrogen fixation capacity and Psolubilization, respectively, under neuter broth (pH 7.0), they wereused to evaluate both abilities under acidic medium (pH 4.0) Theresults showed that all strains have ability of nitrogen fixation, and Psolubilization 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
ASS area Strain
Days after incubation