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Escherichia Coli, Fusarium Proliferatum, F ใบรับรองวิทยานิพนธ์ บัณฑิตวิทยาลัย มหาวิทยาลัยเกษตรศาสตร์ วิทยาศาสตรมหาบัณฑิต (พันธุศาสตร์) ปริญญา พันธุศาสตร์ พันธุศาสตร์ สาขา ภาควิชา เรื่อง การวิเคราะห์ลักษณะและติดตามแอคติโนมัยสีทเอนโดไฟต์ที่คัดแยกจากกระถินณรงค์ (Acacia auriculiformis A. Cunn. ex Benth.) โดยเทคนิคทางโมเลกุล Characterization and Monitoring of Endophytic Actinomycetes Isolated from Wattle tree (Acacia auriculiformis A. Cunn. ex Benth.) Using Molecular Techniques นามผู้วิจัย นายชาคริต บุญอยู่ ได้พิจารณาเห็นชอบโดย อาจารย์ที่ปรึกษาวิทยานิพนธ์หลัก วิทยา ( รองศาสตราจารย์อรินทิพย์ ธรรมชัยพิเนต, Ph.D. ) อาจารย์ที่ปรึกษาวิทยานิพนธ์ร่วม ( ผู้ช่วยศาสตราจารย์วิภา หงษ์ตระกูล, Ph.D. ) หัวหน้าภาควิชา ( รองศาสตราจารย์เลิศลักษณ์ เงินศิริ, Ph.D. ) บัณฑิตวิทยาลัย มหาวิทยาลัยเกษตรศาสตร์รับรองแล้ว ( รองศาสตราจารย์กัญจนา ธีระกุล, D.Agr. ) คณบดีบัณฑิตวิทยาลัย วันที่ เดือน พ.ศ . กกกกก (Acacia auriculiformis A. Cunn. ex Benth.) ก Characterization and Monitoring of Endophytic Actinomycetes Isolated from Wattle tree (Acacia auriculiformis A. Cunn. ex Benth.) Using Molecular Techniques ก () .. 2553 2553: กกกก ก ( Acacia auriculiformis A. Cunn. ex Benth.) ก () กก: , Ph.D. 90 ก 11 กกกกกก ก 16S rRNA ก Streptomyces, Actinoallomurus, Amycolatopsis, Kribbella and Microbispora กก 5 GMKU 932 Bacillus cereus GMKU 937 GMKU 938 Aspergillus niger GMKU 940 B. cereus, Staphylococcus aureus, Escherichia coli, Fusarium proliferatum, F. moniliforme A. niger GMKU 944 B. cereus, S. aureus, Ralstonia solanacearum A. niger egfp Streptomyces sp. GMKU 937 GMKU 944 กก MS MgCl2 10 24 egfp ก Streptomyces sp. GMKU 944 GMKU 937 กกก Streptomyces sp. GMKU 937 GMKU 944/egfp กก Rhizobium sp. ก กก กกก ก Streptomyces sp. GMKU 937 ก GMKU 937 Streptomyces sp. GMKU 944/egfp กกก ก กกกก EGFP Streptomyces sp. GMKU 944/egfp ก confocal laser scanning microscope GMKU 944/egfp ก กก กกก Streptomyces sp. GMKU 944/egfp กกก ก ก Streptomyces sp. GMKU 944/egfp กกก / / กก Chakrit Bunyoo 2010: Characterization and Monitoring of Endophytic Actinomycetes Isolated from Wattle tree (Acacia auriculiformis A. Cunn. ex Benth.) Using Molecular Techniques. Master of Science (Genetics), Major Field: Genetics, Department of Genetics. Thesis Advisor: Associate Professor Arinthip Thamchaipenet, Ph.D. 90 pages. Eleven strains of endophytic actinomycetes were isolated from healthy roots of wattle tree (Acacia auriculiformis A. Cunn. ex Benth.) collected from Bangkok and Nakhonprathom provinces, Thailand. Analysis of 16S rRNA gene sequences of those strains revealed that they belong to members of the genera Streptomyces, Actinoallomurus, Amycolatopsis, Kribbella and Microbispora. Five strains showed antimicrobial activities aginst test microorganisms. Strain GMKU 932 showed activity against Bacillus cereus while strains GMKU 937 and GMKU 938 were active against Aspergillus niger. Strain GMKU 940 showed activity against B. cereus, Staphylococcus aureus, Escherichia coli, Fusarium proliferatum, F. moniliforme and A. niger. Strain GMKU 944 was active against B. cereus, S. aureus, Ralstonia solanacearum and A. niger. The egfp gene was introduced into Streptomyces sp. GMKU 944 and GMKU 937 by intergeneric conjugation and was successfully transfered and expressed in Streptomyces sp. GMKU 944 only. The residing property of both endophytes in the roots of wattle tree was verified by inoculation of Streptomyces sp. GMKU 937 or GMKU 944/egfp alone, and the mixture with Rhizobium sp. to germinated seeds of wattle tree. The results showed that both strains revealed no effect to nodulation. Streptomyces sp. GMKU 937 could be re- isolated from the seedlings, but it was unsuccessful with strain GMKU 944/egfp. However, Streptomyces sp. GMKU 944/egfp could be visualized through EGFP expression by confocal laser scanning microscope and found mainly colonizing at phloem and some at xylem parenchyma cells. SEM micrographs indicated mycelia of Streptomyces sp. GMKU 944/egfp covering root and nodule surfaces. These results suggested that Streptomyces sp. GMKU 944/egfp was closely associated with wattle tree and is a true endophyte. / / Students signature Thesis Advisors signature กกก ก .. กกก .. ก ก กก ก . กก ก ก .. ก กกก . กกกก . ก กก กกกก กกก ก Prof. Dr. Mark Buttner, Department of Molecular Microbiology, John Innes Centre, UK. ก กกก 4503 กก ก ก กก ก ก ก ก. (MRG-WII505S027) ก ก 2553 (1) (1) (2) (3) 1 2 กก 3 กก 18 37 68 ก 70 ก 83 กก ก 89 (2) 1 18 2 19 3 ก E. coli 27 3 กกก 39 4 ก 16S rRNA ก 11 ก ก EzTaxon 42 5 ก 46 6 กกก E. coli ET12567 (pUZ8002/pIJ10257) Streptomyces sp. GMKU 944 55 (3) 1 กก 16S rRNA 7 2 กก 16S rRNA 8 3 pIJ8655 30 4 pIJ10257 32 5 กกกก 38 6 ก phylogenetic tree 11 43 7 กก 50 8 กกก 52 9 กกก 52 10 กก 53 11 กกก 10257seqF 10257seqR 56 12 กก EGFP กก 57 13 กกกกก 8 59 14 กกกกกกก 60 15 กกก confocal laser scanning microscope 62 16 กกกกก Streptomyces sp. GMKU 944/egfp กกก 65 17 กกกกก ก กก 66 18 กกกกก Streptomyces sp. GMKU 944/egfp ก ก กก 67 กกกกก (Acacia auriculiformis A. Cunn. ex Benth.) ก Characterization and Monitoring of Endophytic Actinomycetes Isolated from Wattle tree (Acacia auriculiformis A. Cunn. ex Benth.) Using Molecular Techniques กก ก กก (, 2545) ก กกก Bradyrhizobium ก ก ectomycorrhiza endomycorrhiza กกก ก (Duponnois and Plenchette, 2003; Diuof et al., 2005) กกก ก ก ก กกก กก กก ก ก กก ก กกกกกก กกก กก 16S rRNA กก ก กก enhanced green fluorescent protein (egpf) ก กก กก enhanced green fluorescent protein 1. กกกกก 16S rRNA กก 2. กก ก 3. egfp กก (intergeneric conjugation) กกกกกก ก enhanced green fluorescent protein (EGFP) กก 1. ก กก Class Actinobacteria Subclass Actinobacteridae Order Actinomycetales (Stackebrandt and Rainey, 1997) กก ก ก (hypha) - (substrate mycelium vegetative mycelium) กก (aerial mycelium) ก กก กก ก (single spore) ก Micromonospora Thermononospora ก ก Streptomyces ก (rectiflexible) (retinaculiaperti) ก (spira) กก (verticillati) ก (smooth) (hairy) (spiny) (rugose) ก กกก (sporangia) ก Actinoplanes (Vobis, 1997) กกก กก Nocardioform ก ก Nocardia, Nocardiopsis, Oerskovia, Promicromonospora Intrasporangium กกกกก (cocoid) (rod) ก (nonmotile) ก ก Sporichthya Actinosynnema (planospore zoospore) กกก (flagella) กก กก ก กก ก ก (Vobis, 1997) กกก- ก กกก geosmin (trans-1,10-dimethyl decalol) ก (earth odor) (Williams et al., 1989) 4 กกกก ก Frankia ก กก- ก ก Streptomyces 25-35 6.5-8.0 ก ก ก ก ก 3 ก 13 (Williams et al., 1989) ก กกก (Alexander, 1997) กก Streptomyces ก (secondary metabolite) ก ก กก กก กก กกก (Goodfellow et al., 1988) 2. กกก ก (morphology) กกกกก water agar ก yeast extract ก ก (long-working distance objective lens) ก ก กกก ก ก กกก (scanning electron microscope; SEM) ก กกก ก ก กก ก กก กกกก- กก (synnemata) ก (pseudosporangium) (Williams et al., 1989) 5 ก (cell chemistry) กกกก กก กก ก ก กกก กก (paper chromatography) ก ก diaminopimelic acid (DAP) meso- LL- ก DAP กก กกกก (Lechevalier and Lechevalier, 1970) ก 16S rRNA กกก กกก ก ก 16S rRNA ก ก ก 1940 1950 ก Streptomyces ก ก Streptomyces กก Streptomyces ก กกกกกก ก ก ก ก กก ก 16S rRNA กก (Masakazu et al., 1997) rRNA กก กก 70S ก 2 50S rRNA 23S 5S 3 ก 120 ก 30S rRNA 16S 1.5 ก rRNA 5-10 ก 6 (Lewin, 2000) rRNA S. griseus 16S-23S-5S ก stem and loop 50 (Pernodet et al.,1989) 3′ 16S rRNA 6 5′-CCUCCU-3′ ก ShineDalgano 5′ mRNA ก AUG 3′ major domain ก tRNA A-site P-site กกก initiation factor 3 (IF3) ก (Lewin, 2000) กกก 16S rRNA Streptomyces 158-278 120 กก 20 % (Stackebrandt et al., 1991) ก (alpha region variable region) กก (gamma region) 172-202 ก ก กกกก 1102-1122 ก (beta region) กก กก 5 % ก 16S rRNA ก Streptomyces (Kataoka et al., 1997) 16S rRNA class Actinobacteria order Actinomycetales 30 order phylogenetics กก กกกก (Stackebrandt and Rainey, 1997) 1 2 7 1 กก 16S rRNA (Stackebrandt and Rainey, 1997) 8 2 กก 16S rRNA (Stackebrandt and Rainey, 1997) 9 1. (endophytic microorganism) ก (Azevedo et al., 2000; Strobel, 2003) ก symbiosis mutualism กกกก กก กก ก (opportunistic pathogen) ก mycoplasma achaebacteria ก ก ก (Strobel, 2003) 2. กกกก กกก Frankia ก ก Brunchorst 1886 Frankia กก symbiosis กก (Williams et al., 1989) กกก กก Frankia ก ก กก Streptomyces, Microbispora, Micromonospora Nocardiodes ก 16S rDNA ก nec1 กกก nec1 กก (Coombs and Franco, 2003a) ก ก กกก ก Streptomyces กกก ก กก ก 16S rRNA Terminal restriction fragment length polymorphism (T-RFLP) กกก กก 10 ก (Conn and Franco, 2004) กกก ก Streptomyces กก (Tian et al., 2004) ก ก- ก Streptomyces กก (Cao et al., 2004a; Tan et al., 2006) ก ก (Cao et al., 2004b) ก Kribbella solani (Song et al., 2004) กก ก (Taechowisan et al., 2003) กกกก ก ก กก กกกก กกก (Hasegawa et al., 2006) กก ก (Hasegawa et al., 2006) ก กก ก ก (rhizosphere) กกก 3. 3.1 ก กกกก (Alexander, 1997; Okazaki, 2006) กก กก ก- (Okazaki, 2006) กกก 1978 Hasegawa กกก กก Actinosynnema กกกก ก Agromyces albus กก Androsace sp. 11 (Dorofeeva et al., 2003) Micromonospora coriariae กก Coriaria myrtifolia (Trujillo et al, 2006) Pseudonocardia oroxyli ก Oroxylum indicum (Gu et al., 2006) Micromonospora lupine M. saelicesensis กก Lupinus angustifolius. (Trujillo et al., 2007) ก ก ก Kineococcus gynurae sp. nov. กกกก (Duangmal et al., 2008) Pseudonocardia acaciae sp. nov. กกกก (Duangmal et al., 2009) ก Actinophytocola oryzae gen. nov., sp. nov. (Indananda et al., 2009a) Actinoallomurus oryzae sp. nov. (Indananda et al., in press) 3.
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