[보고서]작물 연관 미생물 유전자의 대용량 기능 분석을 통한 미생물 활성 및 활용 안정성 증진

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보고서 정보

주관연구기관

전북대학교
Chonbuk National University

연구책임자

이용훈

참여연구자

스와나리두타 , 카켐보데이비드 , 와비오나알렉스

보고서유형

최종보고서

발행국가

대한민국

언어

한국어

발행년월

2020-12

과제시작연도

2020

주관부처

농촌진흥청
Rural Development Administration(RDA)

등록번호

TRKO202100009695

과제고유번호

1395062838

사업명

차세대바이오그린21(R&D)

DB 구축일자

2021-09-18

키워드

근권미생물.생육촉진.생물방제.돌연변이.트랜스포존.Rhizomicrobe.Growth promotion.Biocontrol.Mutation.Transposon.

표 Strains and plasmids used in this study
표 Primers used in this study
표 Primers used for taxon-specific qPCR assay targeting 16S rRNA genes for bacteria
표 Number of mutant strains showing variations in antibacterial activity among the transposon disruption mutants of Pseudomonas parafulva JBCS1880
표 Genes identified from Tn mutants of Pseudomonas parafulva JBCS1880 showing changes in antibacterial activity
표 Classification of Tn-inserted genes based on the primary function in the cell
표 Effect of mutation on antagonistic and biocontrol activities of Pseudomonas parafulva JBCS1880. The overlay technique was conducted to test the antagonistic activity of wild-type (WT) and mutants on the growth of Xanthomonas axonopodis pv. glycines. The data represents mean inhibition zone±standard deviation of representative mutants showing range of antibacterial activity as compared to WT. For biocontrol assay, soybean leaves were inoculated with each mutant bacterial suspension and sprayed with the bacterial suspension of Xanthomonas axonopodis pv. glycine. The leaves treated with sterile DW served as control. Data represent percent disease index (%) and vertical bars indicate standard deviation of the means
표 Changes of antibacterial activities in Pseudomonas parafulva JBCS1880 transposon insertion mutants. The overlay technique was conducted to test the antagonistic activity of wild-type (WT) and mutants on the growth of Xanthomonas axonopodis pv. glycines. Photos were taken 5 days after incubation. The indicated wild-type and mutant strains were as follows; WT (JBCS1880), severe loss (>80% reduction compared to wild type: Tn02-F2, Tn13-A5, Tn13-H12, Tn17-E2, Tn44-C1, Tn59-H8); mild loss (50 to 21% reduction: Tn03-F4, Tn20-G9, Tn43-F2), and low loss (<20% reduction: Tn24-G11, Tn74-F12)
표 Control of bacterial pustule by treatment with transposon insertional mutants of Pseudomonas parafulva JBCS1880. The soybean leaves were inoculated with each bacterial suspension and challenged with cell of Xanthomonas axonopodis pv. glycine. The inoculated leaves were observed for symptom development in a plant growth room. The leaves treated with sterile DW served as controls. The picture was taken 7 days after inoculation
표 Correlation of antibacterial activity and other phenotypic characteristics on the overall biocontrol activity of P. parafulva JBCS1880. Results analysis showed a strong positive correlation between (A) antibacterial activity, (B) siderophore production, (C) swarming motility and biocontrol efficacy. A positive but weak correlation was observed between (D) biofilm formation and biocontrol efficacy
표 Effect of mutation on swarming motility and siderophore production of Pseudomonas parafulva JBCS1880. Values that vary from the wild-type level by a statistically significant difference (p<0.05), as determined by Tukey test, are denoted by an asterisk. Data are meansstandard deviation from five measurements in three independent experiments
표 Effect of mutation on swarming motility of Pseudomonas parafulva JBCS1880. Tn mutant colonies were grown on soft LB medium (0.5% agar) for 12 h at 28℃ and changes in swarming pattern recorded. The indicated wild-type and mutant strains were as follows: Wild type, >5% increase: Tn27, 57; >20% increase: Tn94, 189; >50% increase: Tn210, 214. Only representative strains are shown here
표 Effect of mutations on biofilm formation and attachment of Pseudomonas parafulva JBCS1880. Values that vary from the wild-type level by a statistically significant difference (p<0.05), as determined by Tukey test, are denoted by an asterisk. Data are meansstandard deviation from five measurements in three independent experiments
표 Effect of mutations on colony surface architecture. Tn mutant colonies were grown on LB medium for 24 h at 28℃ and changes in colony morphology recorded. Representative strains are shown at here
표 The features of Pseudomonas parafulva JBCS1880 genome
표 Homology of the deduced VILS2 nonribosomal synthetase amino acid sequence from Pseudomonas parafulva JBCS1880
표 Structural sequence alignment of the VlsA protein. The deduced amino acid sequence of VlsA from Pseudomonas parafulva JBCS1880 (PpaJBCS1880) [GenBank: CP031641.1] was blasted with NCBI and compared with those of Pseudomonas fluorescens strain SS101 (PflSS101) [GenBank: EU199080], Pseudomonas putida RW10S2(PpuRW10S2) [GenBank: JN982332.1], Pseudomonas fluorescens SBW25 (PflSBW25) [GenBank: AM181176] and Pseudomonas entomophila L48 (L48) [GenBank: CT573326.1]. The multiple sequences were aligned by CLUSTALW2 program. The identical residues are shown in dark shades while the conservative changes are shown in light shades
표 Structural sequence alignment of the VlsB protein. The deduced amino acid sequence of VlsB from Pseudomonas parafulva JBCS1880 (JBCS1880) [GenBank: CP031641.1] was blasted with NCBI and compared with those of Pseudomonas parafulva CRS01-1 (CRS01) [GenBank: CP009747.1], Pseudomonas putida NX_1 (NX_1) [GenBank: JN982332.1] and Pseudomonas entomophila L48 (L48) [GenBank: CT573326.1]. The multiple sequences were aligned by CLUSTALW2 program. The identical residues are shown in dark shades while the conservative changes are shown in light shades
표 Structural sequence alignment of the VlsC protein. The deduced amino acid sequence of VlsC from Pseudomonas parafulva JBCS1880 (JBCS1880) [GenBank: CP031641.1] was blasted with NCBI and compared with those of Pseudomonas parafulva CRS01-1 (CRS01) [GenBank: CP009747.1], Pseudomonas putida NX_1 (NX_1) [GenBank: JN982332.1] and Pseudomonas entomophila L48 (L48) [GenBank: CT573326.1]. The multiple sequences were aligned by CLUSTALW2 program. The identical residues are shown in dark shades while the conservative changes are shown in light shades
표 Map of the vls gene cluster in Pseudomonas parafulva JBCS1880. The gene clusters encoding VILS2 NRPS domains in vlsA, vlsB, and vlsC are shown as labeled circles: C, condensation; T, thiolation; E, epimerization; TE, thioesterase. The associated regulatory and translocation genes macA, macB, LysE, and LuxR are also indicated. The arrows in vls gene cluster indicate the Tn insertion sites that affected antibacterial activity against Xanthomonas axonopodis pv. glycines and Burkholderia glumae
표 Primary structure of the VILS2 and other viscosin class members of cyclic lipopeptides
표 Induction of a white line in the presence of Pseudomonas tolaasii. The formation of a white line between Pseudomonas parafulva JBCS1880 and P. tolaasii was assayed on King’s B media at 25℃. A white line was formed between P. tolaasii (1) and JBCS1800 (2 and 7), while no line was formed by the strains with mutations in the non-ribosomal peptide synthetases (Tn17-E2, Tn13-H12, Tn78-G10 respectively) (3, 4 and 6). No white line was induced by P. putida JBC17 (5) which was used as a negative control
표 Biocontrol efficacy and antibacterial activity of Pseudomonas parafulva PpaJBCS1880 and its mutants. Soybean plants were inoculated with each bacterial suspension and challenged with cells of Xanthomonas axonopodis pv. glycines (Xag). The severity of bacterial pustule was assessed 3 weeks after challenge inoculation (■). The leaves treated with sterile 0.05 M phosphate buffer served as controls. The inhibition zone against Xag was assessed by overlay inoculation (□). Columns with the same letter are not significantly different (p<0.05) according to LSD
표 Biocontrol efficacy and antibacterial activity of Pseudomonas parafulva PpaJBCS1880 mutants. (A) Soybean plants were inoculated with each bacterial suspension and challenged with cells of Xanthomonas axonopodis pv. glycines (Xag). The inoculated leaves were observed for symptom development in a plant growth room. The photo was taken 3 weeks after inoculation. (B) The overlay technique was conducted to test the antagonistic activity of wild type and mutants on the growth of X. a. pv. glycines. Photos were taken two days after incubation
표 Estimated OTU richness and diversity indices in the phyllosphere
$Comparison of microbial communities using Fast Unifracanalysis. (A) Principal coordinate analysis (PCO) compared the differences between treatments using distance matrix. (B) Dendogram showing distance between treatments calculated based on similarity matrix. Plants treated with PpaJBCS1880 or mutants Tn13-H12 (△vlsA) and Tn03-F4 (△nuoC) were challenged with Xanthomonas axonopodis pv. glycines (Control) and phyllosphere bacterial community in each treatment was analyzed through Illumina MiSeq and data was subjected to CLcommunity software$ 표 Comparison of microbial communities using Fast Unifracanalysis. (A) Principal coordinate analysis (PCO) compared the differences between treatments using distance matrix. (B) Dendogram showing distance between treatments calculated based on similarity matrix. Plants treated with PpaJBCS1880 or mutants Tn13-H12 (△vlsA) and Tn03-F4 (△nuoC) were challenged with Xanthomonas axonopodis pv. glycines (Control) and phyllosphere bacterial community in each treatment was analyzed through Illumina MiSeq and data was subjected to CLcommunity software
표 Relative abundance (%) of bacterial phyla in phyllosphere of soybean plants. Bacterial abundance in the leaves treated with wild type (PpaJBCS1880), mutant Tn13-H12 (△vlsA) and Tn03-F4 (△nuoC), and challenged with Xanthomonas axonopodis pv. glycines (control) were compared. Untreated control was treated with 0.05 M phosphate buffer. The treated leaves were collected and DNA was extracted for Illumina MiSeq sequencing and analysis
표 Relative abundance of major bacterial classes in the phyllospheres of control and treated soybean plants as compared to untreated control. Bacterial community in phyllosphere of wild type (PpaJBCS1880) were compared with treatment of mutant Tn13-H12 (△vlsA) and Tn03-F4 (△nuoC). All the plants were challenged with Xanthomonas axonopodis pv. glycines (control)
표 Relative abundance of major bacterial orders in the phyllospheres of control and treated soybean plants. Bacterial orders were compared between leaves with different treatments of wild type (PpaJBCS1880), mutant Tn13-H12 (△vlsA) and Tn03-F4 (△nuoC), and challenged with Xanthomonas axonopodis pv. glycines (control)
표 Relative abundance of major bacterial families in the phyllospheres of control and treated soybean plants. Bacterial families were compared between leaves with different treatments of wild type (PpaJBCS1880), mutant Tn13-H12 (△vlsA) and Tn03-F4 (△nuoC), and challenged with Xanthomonas axonopodis pv. glycines (control)
표 Relative abundance of bacterial genus on the phyllosphere of soybean plants. Bacterial abundance in the leaves treated with wild type (PpaJBCS1880), mutant Tn13-H12 (△vlsC) and Tn03-F4 (△nuoC), and challenged with Xanthomonas axonopodis pv. glycines (control) were compared
표 Relative abundance of major bacterial species in the phyllospheres of control and treated soybean plants. Bacterial abundance in the leaves treated with wild type (PpaJBCS1880), mutant Tn13-H12 (△vlsA) and Tn03-F4 (△nuoC) were challenged with Xanthomonas axonopodis pv. glycines (control) and compared with untreated control
$Heat map with Unifrac cluster analysis of bacterial genus isolated from the phyllosphere of soybean plants. The cluster analysis displays minimum average ratio of 5% at genus level. Bacterial abundance in the leaves treated with wild type (PpaJBCS1880), mutant Tn13-H12 (△vlsC) and Tn03-F4 (△nuoC), and challenged with Xanthomonas axonopodis pv. glycines (control) were compared. Untreated control was treated with distilled water$ 표 Heat map with Unifrac cluster analysis of bacterial genus isolated from the phyllosphere of soybean plants. The cluster analysis displays minimum average ratio of 5% at genus level. Bacterial abundance in the leaves treated with wild type (PpaJBCS1880), mutant Tn13-H12 (△vlsC) and Tn03-F4 (△nuoC), and challenged with Xanthomonas axonopodis pv. glycines (control) were compared. Untreated control was treated with distilled water
표 Comparative analysis of bacterial abundances. The abundance of Firmicutes, Alphaproteobacteria and Betaproteobacteria in the phyllosphere was assessed by taxon-specific qPCR. Data are presented as the log of gene copies of each bacterial group per gram leaves±SE. Bars with the same letter(s) do not differ significantly at P=0.05 for a given treatment
표 Antagonism of PpaJBCS1880 against different bacteria in plate assay using overlay method. Test plates were prepared with KB agarmixed with cells of each bacterium and PpaJBCS1880 or each mutant strain was spotted on the paper disks placed on the test plates. Inhibition zones was photographed two days post incubation (dpi) at 30°C
표 Influence of cell free culture supernatant of PpaJBCS1880 and its mutant strains on total bacteria on leaves. The wells of 12-well culture plate were filled with 1% PDB (0.6 ml per well) with CFS of PpaJBCS1880,Tn13-H12 (vlsC), orTn03-F4 (nuoC) strains to a final concentration of 1%, 3%, 6%, 12%, 24% and 50%. and phyllosphere microbial cell suspension was then poured in the PTFE cylinder. After 24 h of incubation at 28℃, the suspension was serially diluted and plated in LB medium to estimate the phyllosphere bacteria. Data represent mean log cfu/ml of phyllosphere suspension with standard error. Different letters above the bars indicate significant differences at P = 0.05
표 Population dynamics of Pseudomonas parafulva PpaJBCS1880 and its mutant strains in soybean leaves. Soybean plants were inoculated with each bacterial suspension and the plants were cultivated in a plant growth room. The bacterial populations in the leaves were monitored after 0, 7, 14, and 21 days post inoculation. Bars with the same letter(s) do not differ significantly for respective treatment throughout the time-points at P=0.05. Difference in population of respective mutant as compared to PpaJBCS1880 at a particular time-point was determined using Student’s t-test and *indicate wherever significant difference was observed. Vertical bars indicate (±) standard error
표 Strains and plasmids used in this study
표 Primers used in this study
표 Pseudomonas putida JBC17 transposon insertion mutants identified in changes of germination inhibition compared to wild type
표 The features of Pseudomonas putida JBC17 genome
표 Genes identified from Tn mutants of Pseudomonas putida JBC17 based on changes in inhibition ability of conidia germination
표 Classification of Tn-inserted genes based on the primary function in the cell
표 Inhibition of Penicillium digitatum conidial germination and suppression of green mold of mandarins by treatment with Pseudomonas putida JBC17. For conidial germination assay, conidia of P. digitatum were incubated in 10% PDB with cells of P. putida JBC17 or each mutant separated by PTFE membranes. For biocontrol assay, mandarin fruits inoculated with P. digitatum (106 spores/ml) were treated with bacterial suspensions (1×108 cfu/ml) - and then incubated at 20°C and 90% RH for 7 days. Figure shows representative mutants showing alteration in conidial germination inhibition activity and disease development as compared to wild type
표 Germination of Penicillium digitatum conidia on PTFE membranes in cylinders. Conidia were incubated with Pseudomonas putida JBC17WT and its Tn mutants in 10% potato dextrose broth, and germination percentage (■) and germ tube length (□) were recorded 24 h after incubation at 25ºC. The number of germinated spores from 100 observed in a microscopic view was counted and percentage germination was compared with wild type. The data represent mean±standard deviation of the replications. The same letters are not significantly different at P<0.05 according to the Tukey’s test
표 Comparison of growth of Pseudomonas putida JBC17 and its mutants in nutrient rich and poor media. Growth was assessed in fresh nutrient-limited (10% PDB) and -rich (100% PDB) media were inoculated with each mutant cell (OD600=0.05) and incubated at 28°C with shaking at180 rpm. Growth was compared to that of wild type 12 h after incubation for 100% PDB and 24 h for 10% PDB. The data represent mean±standard deviation of the replications and he same letters are not significantly different at P<0.05 according to the Tukey’s test
표 Comparison of growth of Pseudomonas putida JBC17 and selected mutants in nutrient poor media. The mutants were grown in nutrient-limited medium 10% PDB and high concentration of 50% and 100% PDB. Growth was compared to that of wild type at 12h for 50% and100% PDB and 24h for 10% PDB. Starter cultures (OD600=0.05) were inoculated in fresh media and incubated at 30°C with shaking at 200 rpm
표 Number of carbon substrates utilized by Pseudomonas putida JBC17 and its flagella-related mutants. The number of wells that showing color development by Tn04-B10 (fliH) and Tn04-H1 (fliR) was assayed using Biolog GN2 microplate at 6, 16, 24, and 48 h after incubation and compared to wild type. Data are presented as mean±standard deviation of three replicates and values marked by an asterisk (*) are significantly different at each time point at P<0.05 according to Students’ t-test
표 Average well color development (AWCD) and utilization of various groups of carbon substrates by Pseudomonas putida JBC17WT and flagella-related mutants. AWCD of 95 carbons were recorded as OD600 at 6 h (A), 16 h (B), 24 h (C), and 48 h (D) after incubation using Biolog GN2 microplate. Data are presented as mean ± SD of three replicates, and values marked by an asterisk (*) are significantly different from the wild type in absorbance at P<0.05 according to Students’ t-test
표 Effect of mutations on swimming motility of Pseudomonas putida JBC17 and mutants. Each strain was grown overnight in LB broth and inoculated in the center of 10% and 100% PDA (0.3% agar) plates. The colony diameter was recorded 48 h and 24 h after incubation for 10% and 100% PDA, respectively
표 Effect of mutations on swimming motility of Pseudomonas putida JBC17 and mutants. Each strain was grown overnight in LB broth and inoculated in the center of 10% (A) and 100% PDA (B) containing 0.3% agar, and incubated for 48 h and 24 h, respectively
표 Swarming motility of Pseudomonas putida JBC17 and its Tn mutants. Each strain was grown overnight in LB broth and inoculated in the center of 10% (A) and 100% PDA (B) containing 0.5% agar and incubated for 24 h at 28°C, respectively
표 Complementation of fliR gene into Tn mutant strain Tn04-H1 of Pseudomonas putida JBC17. The gene was cloned into pUCP18Cat and transformed into mutant strain to produce complemented strain (Tn04-H1+fliR). An empty vector (EV) was also transformed into the mutant to produce control strain (Tn04-H1+EV). (A) In vitro growth in 10% and 100% PDB; (B) Swimming motility in (C) 10% and (D) 100% PDA containing 0.3% agar. Vertical bars indicate mean ± SD of the replications. Bars with same letters are not significantly different at P<0.05 according to the Tukey’s test

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작물 연관 미생물 유전자의 대용량 기능 분석을 통한 미생물 활성 및 활용 안정성 증진
Increase of activity and stability of crop associated microorganisms by high-throughput functional analysis of genes contributing plant growth and health 원문보기

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작물 연관 미생물 유전자의 대용량 기능 분석을 통한 미생물 활성 및 활용 안정성 증진 Increase of activity and stability of crop associated microorganisms by high-throughput functional analysis of genes contributing plant growth and health 원문보기

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작물 연관 미생물 유전자의 대용량 기능 분석을 통한 미생물 활성 및 활용 안정성 증진
Increase of activity and stability of crop associated microorganisms by high-throughput functional analysis of genes contributing plant growth and health 원문보기

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