Phân lập, tuyển chọn, nhận diện vi khuẩn nội sinh và vi khuẩn vùng rễ cây mía (saccharum spp l ) trồng trên đất xám tỉnh tây ninh TT TIẾNG ANH

MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY SUMARRY OF DISSERTATION Major: Microbiology Code: 62 42 01 07 POSTGRADUATE NAME: HOANG MINH TAM ISOLATION, SELECTION AND IDENTIFICATION OF ENDOPHYTIC BACTERIA AND RHIZOSPHERIC BACTERIA IN SUGARCANE (Saccharum spp L.) GROWN ON ACRISOLS IN TAY NINH PROVINCE Can Tho, 2021 THE RESEARCH WAS COMPLETED AT CAN THO UNIVERSITY The first supvisor: Prof Dr Cao Ngoc Diep The second supervisor: Assist Dr Nguyen Bao Toan The thesis will be examined by Thesis Defense Committee At: Doctoral Dissertation Defense Auditorium of Can Tho University Time: 8: 00 am; Date: October 27, 14h00 ngày 20 tháng 11 năm 2020 Reviewer 1: Prof Dr Tran Linh Thuoc Reviewer 2: Assist Prof Dr Nguyen Huu Hiep Reviewer 3: Assist Prof Dr Nguyen Van Ba The thesis will be available for reference at: Learning Resource Center of Can Tho University Vietnamese National Library LIST OF PUBLICATION RELATED TO THE THESIS Hoang M.T and N.D Cao, 2017 Isolation and Identification of Rhizospheric Bacteria in Sugarcane (Saccharum spp L.) Cultivated on Acrisols of Tay Ninh Province, Vietnam International Journal of Innovations in Engineering and Technology (IJIET), 8(2): 323-335 Hoang M.T and N.D Cao, 2017 Isolation and characterization of endophytic bacteria isolated from the sugarcane cultivated on acrisols of Tay Ninh province,Vietnam International Journal of Innovations in Engineering and Technology (IJIET), 8(3): 222-236 Hoang M.T , N.T Dang and N.D Cao, 2020 Functional and molecular characterization of plant growth promoting bacteria associated with sugarcane cultivated in Tay Ninh Province, Vietnam GSC Biological and Pharmaceutical Sciences, 2020, 11(02): 265–277 CHAPTER 1: INTRODUCTION Necessity of the research In the sugar industry, sugarcane accounts for over 60% of the world's total sugar production In Vietnam, sugarcane can be grown throughout the country In particular, Tay Ninh is one of the provinces with the largest sugarcane growing areas in Vietnam Most of the sugarcane growing soil in Tay Ninh province belongs to acrisols on ancient alluvial ground with poor nutrition characteristics To maintain high yields, sugarcane growers often use large amounts of chemical fertilizers, especially nitrogenous fertilizers Excessive use of chemical fertilizers as well as plant protection substances not only increases production costs, but also causes to negative effects on plants, the ecosystem, the environment and human health Therefore, the research, development and application of beneficial bacteria, capable of promoting plant growth, and reducing the dependence of sugarcane production on chemical fertilizers are necessary Thus, the doctoral thesis “isolation, selection and identification of endophytic bacteria and rhizospheric bacteria in sugarcane (saccharum spp L.) grown on acrisols in Tay Ninh Province” was carried out to investigate indigenous strains of bacteria capable of supplementing N and P nutrients for sugarcane Objectives The main objectives of the dissertation are to isolate, identify, and select endophyitc and rhizospheric bacteria strains of sugarcane capable of nitrogen fixation, phosphate solubilization that can be used for sugarcane grown in Tay Ninh province New findings and significances of thesis The thesis isolated 422 strains of bacteria with both capacities of nitrogen fixation and phosphate solubilization Of which, there were 36 strains of bacteria with good properties identified by analyzing the 16S rRNA gene sequence The CT4bd isolate (99.76% similarity to Serratia oryzae) combined with TPD3b (96% similarity to Bacillus subtilis) effectively replaced 25% of chemical fertilizers N and P in terms of sugarcane yield and increased 14% CCS when tested on sugarcane grown in the field In particular, the endophytic bacterium strain of sugarcane, CT4bd was identified as Serratia oryzae sp nov., a newly discovered endophytic rice species grown in China and first announced in 2017 by Zhang et al (2017) CHAPTER 2: LITERATURE REVIEWS In general, the sugarcane growing soil in Tay Ninh province is grey exhausted soil Of which, acrisol soil on ancient alluvium base occupies the largest area but is poorest in nutrition The other two types of acrisols are gray soil with gley-patchy layer and gley-gray soil Due to their distribution in low-lying and waterstagnant terrain, both humus and nitrogen content are better In 2018, the average yield of sugarcane in Tay Ninh province reached about 77.18 tons / ha, higher than the average yield of the whole country The sugarcane productivity in Tay Ninh province reached 1,132,011 tons, accounting for 6.7% of the country and accounting for 62% of the Southeast Since most of the land resources of Tay Ninh province are gray soils with low or unbalanced nutrients, in agroforestry on gray soil, it is necessary to invest in fertilizers to achieve high productivity Increasing productivity based on chemical fertilizers will increase production costs, reduce competitiveness in exports, and cause many harms to the environment and human health Thus, the most basic limitation in sugarcane production at present is the problem related to fertilizers, especially chemical fertilizer N, P Therefore, for the goal of sustainable sugar production, Tay Ninh needs to consider measures to increase research and exploit the application of bacteria that promote plant growth associated with sugarcane in order to reduce production costs, increase competitiveness in exports, and at the same time reduce harms to the environment and human health Plant Associated Bacteria (PAB) can be classified into three groups: rihzospheric bacteria, rhizoplanic bacteria and endophytic bacteria depending on the location of residence; or is divided into groups: PAB causes disease and PAB is beneficial depending on their effects on the host For the beneficial PABs group, their primary benefit is their ability to promote growth of the host plant Plant Growth Promoting Bacteria (PGPB) can also be further subdivided into a smaller group, called "PGPB- biocontrol", consisting of probiotics with the ability to increase promotes the health of the plant When PGPBs live in the plant's root zone, they are called "Plant Growth Promoting Rhizobacteria (PGPR)" Mechanism of promoting plant growth can be summarized in the following Schematic diagram (figure 2.1): Figure 2.1: Schematic diagram showing plant growth promoting bacteria affect plant growth directly and indirectly (Gupta et al., 2015) Bacteria associated with sugarcane, are diverse, isolated from rhizospheres, intercellular or endothelium of sugarcane plants grown in many countries around the world These bacteria belong to the genera: Azospirillum, Azotobacter, Herbaspirillum, Bacillus, Burkholderia, Pseudomonas, Rhizobium, Gluconacetobacter… Currently, there has been more and more new reports about plant growth-promoting bacteria associated with sugarcane grown in countries around the world and in the neibouring regions such as China and Thailand, but most of these publications did not specify the composition of the sampled or tested soil On the basic research situation on bacteria associated with sugarcane grown on acrisols, there are a number of publications related to the topic, mainly from Brazil and Vietnam Bacteria that promote plant growth associated with sugarcane grown on acrisols in Brazil are common in the genus Pantoea, Burkholderia, Pseudomonas, Enterobacter, and Klebsiella The results of density analysis of the above study showed that the number of bacteria in the rhizospherie soil was 20 times higher than that inside the root The density of root’s endophytic bacteria was thousands of times higher than that of stem’s endophytic bacteria In Vietnam, the results of two studies to isolate phosphate solubilization and nitrogen fixation bacteria in acrisols in Dong Nai province showed that these bacteria belonged to the genuses: Bacillus, Sphingomonas, Raoultella, Achromobacter and Klebsiella Methods of studying associated bacteria (endophytic and rhizopheric bacteria) with sugarcane include isolation, characterization and identification Depending on interaction of the bacteria with the host plant, there are different sample handling methods Media for isolation and evaluation of nitrogen fixing ability for nitrogen bafixation bacteria are liquid nitrogen-free, semi-solid or solid medium such as NFb and JNFb, LGI and LGI-P, JMV, Baz and Bac, Burk's Media for evaluating the phosphate solubilization of nitrogen-fixing bacteria are insoluble phosphate containing media such as Pikovskaya, NBRIP, NBRIPBPB, and GELP Determination of the ability to stimulate plant growth of bacteria through quantitative analysises of N, IAA and P2O5 content generated in culture media by colorimetric spectroscopy method Identification of nitrogen fixation and phosphate solubilization bacteria has been based on traditional methods such as Gram's method, morphology and molecular biology method using 16SRNA gene marker Researches on the plant growth-promoting effects of sugarcane associated bacteria have been mainly in three forms: (1) using bacteria isolated from sugarcane to inoculate back to sugarcane, (2) use bacteria isolated from other crops to inoculate sugarcane, (3) use bacteria isolated from sugarcane to inoculate other crops However, this section only covers documentation relating to form (1) and (2) The results of these trials showed that bacteria used alone or in combination had the ability to stimulate growth, resistance to disease, increase in fresh biomass, dry biomass, increase yield in sugarcane, and reduce significantly chemical fertilizers used in sugar cane production The effect of plant growthpromoting bacteria was believed to be most effective when the bacteria isolated from the sugarcane plant were reintroduced to sugarcane grown in native soil Objects inoculated with plant growth promotion bacteria are sugarcane cuttings or micropropagated The density of nitrogen fixation bacteria in soil samples was quite high in the range of lg CFU / g of soil from 5.74 to 9.37, corresponding to about x 105 - 2.2 x 109 CFU / g of soil The average nitrogen fixation bacteria density of about lg CFU / g of soil was 6.53, corresponding to 3.4 x10 CFU / g of soil The density of phosphate solubilization bacteria ranged from lg CFU / g of soil from 5.17 to 9.72, corresponding to about 1.6 x 10 - x 109 CFU / g soil On average, the phosphate solubilization bacteria density was lower than that of nitrogenfixation bacteria, about lg CFU / g of soil was 6.45, equivalent to 2.8 x106 CFU / g of soil This result was higher than the published density of groups of nitrogen-fixing bacteria and phosphate solubilization bacteria in acrisols of growing sugarcane in Dong Nai province (lg CFU / g of soil is from 5.01 - 8,18) (Hoang and Cao, 2014) The linear relationship between nitrogen fixation bacteria population and phosphate solubilization bacteria and soil pHH2O was statistically insignificant Meanwhile, the correlation and linear regression between these two bacteria and the organic matter content in the soil was statistically significant at the alpha level of 0.01 Especially, there was a strong correlation between the content of organic matter and the density of solubilization bacteria ((Y = 0.7247x + 4.8158; R=0.765)) This is the 21 scientific basis for the detection and application of beneficial soil microbes in coordination with organic material sources in bio-fertilizer production for plants 4.2 Results of isolation of rhizospheric and endophytic bacteria of sugarcanes grown in acrisols in Tay Ninh province In total, the thesis isolated 422 strains of bacteria associated with sugarcanes with the first two basic plant growth-promoting properties of nitrogen fixation and phosphate solubilization Of which, there are 246 strains of rhizospheric bacteria and 176 strains of endophytic bacteria There were 109 strains of bacteria isolated on NBRIP medium (accounting for 25.8%), 137 strains isolated on Burk's medium (accounting for 32.5%) and 176 strains isolated on LGI medium (accounting for 41.7%) 4.3 Morphology of rhizospheric and endophytic bacteria cell and colony of sugarcanes grown in acrisols in Tay Ninh province The main colors of colonies of bacteria isolates when growing on the isolation medium were opaque white (37.0 - 45.5%) and light yellow (40.2 - 46.0%) The colonies were circular about 85.8 - 92.0%, with the whole cover about 72.8 - 89.8% and with the elevate about 0.9 - 93.5%) (Figure 1A) In terms of colony diameter, most colonies were very small There were 22 more than 70% of colonies of strains less than mm in diameter (Figure 4.1B) Bars in figure 4.3 B with the length of 150 µm Figure 4.1: Morphology of colonies (A) and colony diameters (B) of some rhizospheric and endophytic bacteria 4.4 Results of quanlication of capacities of nitrogen fixation, phosphate solubilization and IAA production in vitro of rhizospheric and endophytic bacteria of sugarcane grown in Tay Ninh province As for the nitrogen fixing ability of the rhizospheric bacteria, 46 out of 246 isolates had good quantitative results; of which, 19 isolates achieved average NH 4+ level ≥1 mg / L The best isolate was TPD3d reached 2.45 mg / L NH 4+ For phosphate solubilization there 23 were 64 strains showing good quantitative results; of which 19 strains achieved average P O5 level of equal or greater than 200 mg / L The best strain was TAC4a reaching 344.68 mg / L P O5 For the ability to produce IAA, only 60 isolates out of 246 strains of rhizospheric bacteria demonstrated the ability to produce IAA in the absence of tryptophan (30.5%) Of which, 40 strains had average IAA levels ≥5 mg / L The best strain was BCA37 reaching 12.1 mg / L IAA Based on the results of NH 4+, P2O5 and IAA measurements, 30 strains of rhizospheric bacteria were selected for the PCR experiments and 16S rRNA gene sequencing As for the nitrogen fixing ability of endophytic bacteria, 44 out of 176 strains showed good quantitative results; of which, 10 lines achieved average NH4 + level ≥1 mg / L The best strain was CT4bd and reached 1.94 mg / L NH + For phosphate solubilization, there were 56 strains showing good quantitative results; of which 22 strains achieved average P2 O5 level ≥200 mg / L The best strain was CR4c and reached 311.63 mg / L P 2O5 As for the ability to produce IAA, out of 176 endophytic strains, only 41 isolates showed the ability to produce IAA in the absence of tryptophan (23.3%) Of which, 19 isolates had average IAA levels ≥20 mg / L The best strain was TAC4a and reached 68 mg / L IAA 24 Based on the results of NH 4+, P2 O5 and IAA quantity analyses, 36 endophytic bacterial strains were selected for PCR experiments and 16S rRNA gene sequencing 4.5 Results of qualitative analysis of capacities of siderophore production of rhizospheric and endophytic bacteria of sugarcanes grown in Tay Ninh province Research results show that out of 422 isolates, there are 44 strains of rhizospheric bacteria and endophytic bacteria with capable of producing siderophore through liquid CAS test, accounting for 10.4% The color of the reaction between the siderophore and the CAS reagent was also quite diverse as described by some previous authors (Sullivan et al., 2012; Thanh and Tram, 2018) (Figure 4.2) 25 Figure 4.2: Capacities of producing siderophore of some rhizospheric and endophytic bacteria of sugarcanes grown in Tay Ninh province through liquid CAS test As for the ability to develop and create a halo ring on a solid CAS medium, only 38 strains showed this ability (accounting for 9.0%) Some strains have quite large halo diameter and bacteria could grow on CAS agar medium such as TBR3a, DMC5a, GR3; but there are also some strains that grew in the medium without forming a halo ring as described previously by Chaitanya et al (2014) (Figure 4.3) 26 Bars in figure 4.3 with the length of cm Figure 4.3: Capacities of producing halo rings of some rhizospheric and endophytic bacteria of sugarcanes grown in Tay Ninh province through solid CAS test 4.6 Results of identification of some selected rhizospheric and endophytic bacteria of sugarcanes The results of electrophoresis of PCR products of 16S rRNA gene sequence with primers 8F and 1492R for rhizospheric bacteria and with a primer pair p515FPL and p13B for endophytic bacteria produced bands of 1,500 bp and 900 bp (Figure 4.4 and Figure 4.5) M: 100 bp PLUS™ DNA Ladder (GoldBio); C: Negative control; – 14: PCR products of BC12, BC37, CHT2f, GOD1c, GOD2f, DMC2a, DMC2c, DMC5e, TAB01, TPD3b, TAC4a, TCD2b, TDB2e, TRB1b Figure 4.4: Bands of 1.500 bp of PCR products of rDNA sequences with a primer 8F and 1492R for rhizospheric bacteria 27 M: 100 Bp Dna Ladder (Thomas Scientific); C: negative control – 10: PCR products of BCR5a, CT4bd, MR4c, GT1e, GT3b, GR3, TPR4b, TCT1a, TR3a, TBR3a Figure 4.5: Bands of 900 bp of PCR products of 16S rDNA sequences with a pair of p515FPL and p13B for endophytic bacteria There are 17 strains of rhizospheric bacteria and 19 strains of endophytic bacteria ere identified on the basis of 16S rDNA sequence homology and phylogenetic tree, cell shape, Gram staining results and scanning electron microscopy The bacterial strains have been identified including 17 rhizospheric strains and 19 strains of endophytic bacteria, belonging to the genera: Enterobacter, Burkholderia, Bacillus, Stenotrophomonas, Kosakonia, Serratia, Advenella, Paraburkholderia, Chitinophaga, Herbaspirillum, and Acinetobacter The genera: Enterobacter, Burkholderia and Bacillus were present in both rhizospheric and endophytic bacteria In which, 28 genera Enterobacter, Burkholderia and Bacillus were present in both subjects; Bacillus in the rhizophere (35%) was more abundant than that in the endothelium (16%) (Figure 4.6) The putative names of 36 species are presented in Tables 4.8 and 4.9 of the thesis Figure 4.6: Ratios of homologous genera of 17 rhizopheric isolates and 19 endophytic isolates when identified 4.7 Results of evaluation of plant growth promotion effects of rhizospheric and endophytic bacteria on in vitro sugarcane seedlings grown in the net house In vitro seedlings were produced by micropropagation from apical tips on the best MS medium supplemented with 1.5 mg / L BA at the shoot-forming stage, supplemented with mg / L BA and 0.5 mg / L Kin for the shoot multiplication phase, supplemented 0.25 mg / L NAA for the rooting and forming stage of complete seedlings Twelve strains of bacteria with good capacities to nitrogen fixation, phosphate solubilization, and IAA synthesis and siderophore production were selected to continue to be 29 evaluated for their plant growth promoting properties in sugarcane seedlings grown in Leonard jars under the net house’s conditions after 90 days of planting (Table 4.16 in the thesis) The results showed that the two strains of bacteria with the best efficiency on most evaluation criteria were soil bacteria TPD3b (Bacillus subtilis) and endogenous bacteria CT4bd (Serratia oryzae) The endophytic strain CT4bd was originally isolated from sugar cane stalks grown in Hao Duoc commune, Chau Thanh district and the bacterium TPD3b was isolated from the rhizosphere of sugarcane roots grown in Tan Phong commune, Tan Bien district, Tay Ninh province The effects of strains of bacteria TPD3b and CT4bd on growth criteria of sugarcane cultured tissue grown under net house conditions are summarized in (Table 4.1) Table 4.1: Summary of efficiency of two bacteria strains TPD3b and CCT4bd on in vitro seedlings’ growth on the net house condition Criteria Root number Leaf number Stem height (cm) Root’s fresh biomass (g) Stem’s fresh biomass (g) Leaf’s fresh biomass (g) Whole sugarcane biomass (g) Root’s dry biomass (g) Stem’s dry biomass (g) Leaf’s’s dry biomass (g) Whole sugarcane’s dry biomass (g) TPD3b (Bacillus subtilis) 24,00 b ± 0,82 5,75 ef ± 0,50 59,00 a ± 1,32 1,58 a ± 0,03 0,54 a ± 0,03 0,71 b ± 0,05 2,83 a ± 0,09 0,33 a ± 0,02 0,19 a ± 0,01 0,08 b ± 0,01 0,17 b ± 0,01 30 Results CT4bd (Serratia oryzae) 29,75 a ± 3,50 6,75 cd ± 0,50 55,50 b ± 1,47 0,95 b ± 0,13 0,40 b ± 0,01 1,02 a ± 0,09 2,37 b ± 0,19 0,26 b ± 0,03 0,08 b ± 0,01 0,72 a ± 0,01 0,50 b ± 0,04 4.8 Results of capacities of plant growth promotion of selected rhizospheric and endophytic bacteria on the potted sugarcanes Two strains CT4bd (Serratia oryzae) and TPD3b (Bacillus subtilis) with the best results in section 4.7 were evaluated for their ability to promote plant growth on sugarcane variety K95-156 grown in pots when applied individually or together with chemical fertilizer N and P at different fertilizer rates Through the results of testing the plant growthpromoting effects of selected strains CT4bd and TPD3b on sugarcane variety K95-156 after six months of planting, the following summary trends can be drawn: (1) In the absence of using chemical fertilizers, both strains of bacteria had good effects on all parameters compared to the treatment without using bacteria (2) The fertilization level of 25% N and P fertilizers in combination with bacteria did not have a significant effect on the growth and yield of sugarcane, so these treatments could be reduced in the field experiment later (3) CT4bd strain, when used in combination with N, P fertilizer, increased stem diameter, Brix degree and commercial cane sugar (4) The TPD3b isolate when used in combination with N, P fertilizer increased the number of internodes, Brix degree and commercial cane sugar (5) The treatments used bacterial strains in combination with 75% N and 31 P fertilizer had the best effect on all monitoring criteria Thus, two strains CT4bd and TPD3b used individually or in combination continue to be studied for the effect on growth and yield of sugarcane variety K95-156 in combination with chemical fertilizers N and P at 0%, 50%, 75% and 100% 4.9 Results of capacities of plant growth promotion of selected rhizospheric and endophytic bacteria on sugarcanes grown in the field Two strains of bacteria CT4bd (Serratia oryzae) and TPD3b (Bacillus subtilis) were continuously evaluated for their abilities to promote plant growth in field sugarcane when applied alone or in combination with chemical N and P fertilizers at different levels of fertilization For sugarcane varieties K95-156 grown on acrisols in Tay Ninh province, both selected bacterial strains CT4bd (Serratia oryzae) and TPD3b (Bacillus subtilis) showed capacities of promoting sugarcanes’ growth in the field Specifically: (1) The strain CT4bd combined with 75 - 100% N, P fertilizer had good performance on criteria including internode number, actual cane yield, Brix degree, commercial cane sugar and sugar yield (2) The strain of TPD3b bacteria combined with 75 100% of N, P fertilizers had a good effect on 32 parameters including internode number, Brix degree, commercial cane sugar and actual sugar yield; (3) The combination of strains CT4bd and TPD3b combined with 75% fertilizer N, P had good effect on criteria of internode index, Brix degree, commercial cane sugar and actual sugar yield (Table 4.2) Table 4.2: Summary of some results of strains CT4bd and TPD3b combined with the fertilizer levels on the growth and yield indicators of sugarcane K95-156 after 12 months of planting in the field internode number Stem height Yield Brix CCS Sugar yield CT4bd TPD3b CT4bd TPD3b (streatment B1F-) (streatment B2F-) 4,24% (B1F2) 4,24% (B2F3) 8,4% (B3F2) 1,97% (B1F2) 2,54% (B1F2) 9,45% (B1F2) 5,58% (B2F3) 7,43% (B2F3) 7,3% (B3F2) 9,8% (B3F2) 14,4% (B3F2) (streatment B3F-) The data presented in the Table are the percentage increase of each treatment compared with the positive control B0F4 without bacteria strains and 100% of chemical fertilizer N, P Streatment notation: (F0): 0% NP; (F1): 50% NP; (F2): 75% NP; (F3): 100% NP ; (B0): VK; (B1): Serratia oryzae CT4bd; (B2): Bacillus subtilis TPD3b; (B3): Serratia oryzae CT4bd + Bacillus subtilis TPD3b Thus, the best treatment proposed for larger-scale experiments was the combination of two strains 33 CT4bd and TPD3b combined with 75% fertilizer N, P This experiment saved 25% of chemical fertilizer N, P and increased 8.4% in the internode number; 7.3% in Brix; 9.8% in CCS; and 14.4% in actual sugar yield 4.10 Analysis of nutrient composition of the postharvest soil After the sugarcane was harvested, the nutrient content of each treatment was analyzed The results showed that the use of bacteria had a positive impact on the N, total organic matter and phosphorus content that is readily soluble in the soil In which, bacteria CT4bd or bacteria combination CT4bd and TPD3b had a good effect on the total N and soluble P components of the soil after harvest; while TPD3b bacteria was most effective in improving the total organic matter composition of the soil after harvest 34 CHAPTER 5: CONCLUSIONS AND RECOMMENDATIONS The thesis isolated 246 strains of rhizospheric bacteria and 176 isolates of endophytic bacteria had both capacities of nitrogen fixation and phosphate solubilization with average NH4 + content ranged from to 2,454 mg / L; and average soluble phosphate content measured from 50 to 300.52 mg P2O5 / L There are 101 strains of bacteria capable of synthesizing IAA and 81 strains of bacteria capable of synthesizing siderophore Two selected isolates CT4bd (Serratia oryzae CT4bd) and TPD3b (Bacillus subtilis TPD3b) when combined each other and along with 75% of fertilizer N, P produced sugarcane yield exceeding 4.6% and sugar yield exceeding 14.4 - 21.7% compared to that of The treatment with 100% of N, P fertilizer and no use of bacteria These two strains are proposed to further evaluate the growth promoting effects of sugarcane on different sugarcane varieties grown in Tay Ninh province in both plant-cane and ratoon crops in different raw material sugarcane areas of Tay Ninh province 35 ... announcement of Natural Institute of Agricultural Planning and Projection (200 4) of Vietnam on the acidic properties of Tay Ninh' s acrisols: gley gray soil and gray soil on ancient alluvial with pHH2O... BASE Laboratories Sdn Bhd (Malaysia) The bioinformatics tools used for specifying species names and building phylogeny trees include NCBI's BLASTn (nucleotide BLAST) and MEGA X (Molecular Evolution... al, 2003; Tran Linh Thuoc et al, 200 1) 3.6 Determination of colony morphology and cellular shape and mobility of bacteria isolates 12 Determined properties of colony morphology were size, color,