臺灣博碩士論文加值系統

水稻是世界上多數人口的主要糧食作物，90%以上的水稻是由亞洲地區人口食用。植物寄生性線蟲是限制水稻生長的眾多重要因子中的一項，最高可造成90%以上的產量損失。此研究在台灣地區進行水稻田中根瘤線蟲的種類調查，總共收集了8個線蟲族群，1個族群(TRS) 來自桃園改良場，2個從楊梅地區(YM-TK14, YM-Tn11)，大園(DY)、新屋(XW)、中壢(ZL)地區則各採集1個族群，崙背則有2個族群(LBC, LBF)。這8個族群皆建立了單一母蟲品系，並培養於台南11號水稻上。此研究利用了陰門膜紋，型態，二齡幼蟲體長，DNA序列及同功異構酶圖譜這幾項特性將根瘤線蟲的種類鑑定出來。這8個族群的陰門膜紋之背側因尾部較突出而呈現脊狀壓紋，整體呈卵型近圓形，背側拱型不高。8個族群的個體多與M. graminicola已發表之膜紋相似，少數個體則較類似M. oryze與M. trifoliophila。此8個族群所測量之二齡幼蟲體長介於225.97 – 285.11μm，與相近的M. oryze與M. trifoliophila相比明顯較短。這8個族群利用核酸增幅技術皆得到1個約531bp大小的粒線體COΠ基因條帶，每個族群皆送3 – 5條序列進行定序。這些序列與NCBI資料庫已知的兩個M. graminicola序列( 編號GU187309 , JN241927) 比對有98 – 100%的相似度。以特定探針增幅，此8個族群皆得到764bp大小的核醣體D2/D3序列，解序後與NCBI資料庫比對，發現和已知M. graminicola ( 編號 HQ420904) 序列有99 – 100%相似度。本研究的8個水稻根瘤線蟲族群皆具有相同的同功異構酶圖譜；它們都具有VS1型酯酶，N1型的穀氨酸轉草醯乙酸酶及H1型的超氧化物歧化酶，皆與M. graminicola文獻所記載相同。本研究為第一篇利用多種方式鑑定台灣地區水稻上發現的根瘤線蟲種類。這些結果可增加現有的根瘤線蟲資料庫，並且確認台灣水稻地區所發生的根瘤線蟲種類。

Rice (Oryza sativa) is an important staple food crop for the majority of human population in the world and particularly in Asia. More than 90% of the world rice is grown and consumed in Asia. Among various pests and diseases which constitute important constraints in the successful crop production, plant parasitic nematodes play an important role and account for yield losses to the extent of 90%. This study conducted a survey on the rice growing areas in Taiwan, a total of 8 root-knot nematode isolates were collected, one isolate was collected from Taoyuan District Agricultural Research and Extension Station (TRS), 2 isolates (YM-Tk14 and YM-Tn11) from District Yangmei, from Dayuan, Zhongli and Xinwu each collected one isolates and Lunbei had 2 (LBC and LBF). The single egg mass lines were established from these isolates and maintained on rice cv. Tainan11. The isolates were characterized using female perineal patterns, second-stage larvae body length, DNA sequence analyses and isozyme profiles in order to ascertain their identity. The perineal patterns from these isolates were dorsal ventral elongated with prominent ridges, oval to almost circular in shape and moderate in the height of arc. The patterns of the 8 isolates were similar to M. graminicola with some minor variations and overlap with M. oryzae and M. trifoliophila. The morphology of second-stage larvae was typical for the genus. It was distinguished by the second-stage larvae 225.97-285.11 μm long. It differs from closest relative M. oryzae and M. trifoliophila by shorter second-stage larvae. The mitochondrial DNA region (mtDNA) of the cytochrome oxidase subunit II (COII) of eight isolates all resulted a 531 bp product and each isolates have 3-5 clones sequenced. When blast the sequences with NCBI (Accession GU187309 and JN241927), they were 98-100% identical to M. graminicola. The 28S D2/D3 expansion segments within the ribosomal DNA (rDNA) of eight isolates all resulted a 764 bp product and each isolates had 3-5 clones sequenced, they were 99-100% identical to M. graminicola (Accession HQ420904) from NCBI. All 8 isolates had uniform phenotypes which was the VS1 esterase type, N1 GOT type and H1 SOD type, these phenotypes were typical for M. graminicola. Multienzyme phenotypes often offered biochemical profiles more valuable for definitive characterization of Meloidogyne species than single enzymes. This is the first study of identifying rice root-knot nematodes in Taiwan using multiple approaches. The results will contribute more information of Meloidogyne genus to the current database, and helped us to confirm the RKN nematode species associated with rice in Taiwan.

I. Introduction 1
II. Material and Methods
1. Surveying and Collecting nematode 5
2. Establishing single-female lines 5
3. The morphology of perineal patterns female 6
4. The morphology of second-stage juveniles body length 6
5. Identification by different DNA regions
5.1 Genomic DNA extraction 7
5.2 Amplification of DNA template and purification 8
5.3 Purification of PCR products 9
5.4 Ligation and transformation 9
5.5 Sequencing and data analysis 10
6. Isozyme phenotypes analysis 11
III. Results
1. Surveying and Collecting nematode 15
2. The morphology of mature female 15
3. The morphology of second-stage juveniles body length 16
4. Identification using DNA sequence 17
5. Isozyme phenotypes 19
IV. Discussion 20
V. References 26
VI. Tables 33

VII. Figures 39
VIII. Appendix table 52

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