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研究生:柏圖妮
研究生(外文):Nxumalo Pertunia
論文名稱:以原生植物生長促進菌與植生粒劑促進相思樹於崩塌地之植生復育
論文名稱(外文):Maximizing Growth of Acacia confusa through Native Plant Growth Promoting Bacteria Inoculation and Seed Pelleting for Revegetation in Landslide areas
指導教授:趙國容趙國容引用關係陳玟瑾陳玟瑾引用關係
指導教授(外文):Kuo-Jung ChaoWen-Ching Chen
口試委員:簡士濠
口試委員(外文):Shih-Hao Jien
口試日期:2021-07-21
學位類別:碩士
校院名稱:國立中興大學
系所名稱:國際農學碩士學位學程
學門:農業科學學門
學類:一般農業學類
論文種類:學術論文
論文出版年:2021
畢業學年度:109
語文別:英文
論文頁數:107
中文關鍵詞:相思樹細菌接種植物生長促進細菌植生復育種子顆粒
外文關鍵詞:Acacia confusaBacteria inoculationPlant growth promoting bacteriaRevegetationSeed pellets
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相思樹是一種具有多功能性質且生長迅速的豆科含羞草亞科金合歡屬植物。原產於台灣南部的相思樹是推薦用於崩塌地區的重新造林。然而,在崩塌地種植種子和幼苗並不容易,崩塌地散佈於廣闊的地形上並且相思樹的種子具有休眠的需求。本研究旨在通過種子預處理和接種天然細菌來控制發芽率和生長。此研究將生長於大肚台地上的相思樹根圈和內根圈分離出54種天然細菌。這些分離菌種於培養皿中分別測試是否具有促進或抑制相思樹種子生長的能力。經過培養皿測試,顯示出顯著結果的 16 個分離菌株則進行室外盆栽實驗。此實驗透過測量樹苗枝條和根的長度、芽和根的生物質量、葉葉綠素含量、樹葉發育及植物存活率評估菌株促進生長的效果。分離出的16種菌株則進行植物生長促進 (PGP) 特性的評估,結果顯示使用濃硫酸浸泡20分鐘後可減少種子的休眠時期,並提高92% 種子發芽率,而控制組則是14% 種子發芽率。雖然濃硫酸的處理顯示出最好的結果,但是大量處理並不容易。因此,濃硫酸處理法建議使用作為相思樹育苗的預處理。由於研究方法需要一個簡單、容易並且可以在短時間內完成大量種子的浸泡處理,我們使用100 °C 高溫浸水處理於此項研究中,此處理顯示出高達 78% 的發芽率。16S rDNA 基因定序方法用於系統對照,結果顯示16種菌株中大部分屬於芽孢桿菌屬。而這些菌株對一種或多種 PGP 性狀呈陽性。菌株接種在培養皿中尤其為預處理相思樹種子的測試中,皆為具有顯著的促進或抑制相思樹種子萌發和生長的能力。透過盆栽實驗得知接種菌株 A2TP3 Bacillus proteolyticus 以及 A2SP5 Lysinibacillus sphaericus 具有顯著促進幼苗生長和樹葉發育性能。此兩種菌株則用於進一步用於種子顆粒的製備材料並且進行評估。結果顯示在種子顆粒中接種菌株會抑制種子發芽。然而,種子的發芽率會根據所使用的不同混合物而有不同的結果並沒有明顯的趨勢。該研究表明,細菌接種被證明是盆栽相思樹生長具有前途的生物接種劑。進一步的研究還可以進一步測試在崩塌地接種天然細菌的效果。
Acacia confusa is a fast-growing, multipurpose leguminous shrub native in Taiwan that is recommended for reforestation of landslide areas. However, planting seeds/seedlings in landslide areas is difficult as landslides scatter over a wide terrain and Acacia species faces seed dormancy. Therefore, this study aimed to manipulate germination rate and growth through seed pretreatment and inoculation with native bacteria. A total of 54 native bacteria were isolated from the rhizosphere and endo-rhizosphere of A. confusa growing in the Dadu plateau. Isolates were then tested for their ability to stimulate or inhibit the growth of Acacia confusa seeds on petri dishes. The 16 isolates that showed significant results in the petri dish were then evaluated in a pot test by measuring the shoot and root length; shoot and root biomass, leaf development, leaf chlorophyll content and plant survival. These isolates were also evaluated for their plant growth promoting (PGP) traits. Use of concentrated H₂SO₄ for 20 min, reduced seed dormancy, and enhanced seed germination up to 92%, compared with 14% for the control. Although using H₂SO₄ showed the best result, but it is not easy to handle with mass quantity. Therefore, in this study we used the 100 0C water treatment which showed a germination of up to 78% since it simple, easy and can be done in short space of time for large number of seeds. Concentrated H₂SO₄ is therefore recommended as a pretreatment for nursery seedling of A. confusa. The 16S rDNA gene sequencing method was used for phylogenetic comparison; most of the 16 strains belonged to the Bacillus genus. Bacteria strains were positive for one or more of the PGP traits. Bacteria inoculation either significantly inhibited or improved seed germination and growth especially of pretreated seeds on petri dishes. Seedling growth parameters were significantly improved by inoculation on the pots with isolates A2TP3 Bacillus proteolyticus and A2SP5 Lysinibacillus sphaericus exhibiting strong abilities to improve shoot and leaf growth were selected for further evaluations in seed pellet making. Inoculation in seed pellets inhibited seed germination. However, germination varied according to the different mixtures used. This study demonstrated that bacteria inoculation proved to be promising bio inoculant for A. confusa in pots. Further studies can also test the effect of native bacteria inoculation in landslide areas.
Abstract i
Table of contents iii
List of Tables vi
List of Figures vii
List of Appendices ix
1. Introduction 1
1.2 Objectives 5
1.2.1 Specific objectives 5
2. Materials and Method 6
2.1 Study framework 6
2.2 Pretreatment of seeds- in vitro germination 7
2.2.1 Experimental design 7
2.2.2 Germination parameters measured 9
2.3 Soil and root nodules collection 9
2.4 Bacterial isolation and purification 10
2.4.1 Roots and roots nodules endophytes isolation 10
2.4.2 Soil rhizobacteria isolation 11
2.5 Testing efficacy of isolated bacteria on germination and growth of A. confusa 12
2.5.1 Seed inoculation and germination-petri dish trial 12
2.5.2 Pot culture experiment to evaluate the efficacy of bacteria isolates on A. confusa 13
2.5.2.1 Measurement of leaf chlorophyll index 15
2.6 Seed pellet preparation 16
2.6.1 Acquisition of bulking materials 16
2.6.2 Experiment design 17
2.7 DNA extraction, PCR program, and molecular identification 18
2.8 Plant growth promoting (PGP) traits of the isolated bacterial strains 19
2.8.1 Phosphate solubilizing ability test 19
2.8.2 Indole Acetic Acid (IAA) production capability test 20
2.8.3 Nitrogen-fixing ability test 20
2.8.4 Hydrogen cyanide (HCN) production 21
2.8.5 Screening of siderophore producing bacterial isolates 22
2.9 Statistical analysis 22
3. Results 24
3.1 Effect of A. confusa seed pretreatment on germination 24
3.1.1 Germination percentage 24
3.1.2 Mean germination time and germination index 24
3.2 Bacteria isolation and identification 25
3.3 Characterization of plant growth promotion (PGP) traits 27
3.4 Effect of bacteria inoculation on A. confusa seeds (petri dish experiment) 29
3.5 Effect of bacteria inoculation on growth of A. confusa seedlings -pot experiment 32
3.5.1 Effect on shoot length 32
3.5.2 Root length and volume 35
3.5.3 Leaf (phyllodes) growth 39
3.5.4 Monitoring chlorophyll content 41
3.5.5 Shoot and root biomass 45
3.5.6 Survival 50
3.6 Seed pelleting 52
3.6.1 Effect of bacteria inoculation on A. confusa seed germination in seed pellets 52
4. Discussion 55
4.1 Seed pretreatment 55
4.2 Bacteria isolation and characterization 57
4.3 Effect of native bacteria inoculation on A. confusa germination and growth 59
5. Conclusion 65
6. References 67
7. List of tables 83
8. List of Figures 86
9. List of Appendixes 87
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