臺灣博碩士論文加值系統

本研究自7種芭菲爾鞋蘭根部分離出8株菌種，分別為Pwar-1、Peme-1、Ppri-1、Phen-1、Phel-1、Pdel-1、Pdel-2及Pmic-1，所有菌種經Acridine orange螢光染劑染色後，Pwar-1為多核菌種，Peme-1、Ppri-1、Phen-1、Phel-1、Pdel-1、Pdel-2及Pmic-1為雙核菌種。Peme-1、Phen-1、Phel-1、Pdel-1、Pmic-1菌種於WA培養基內會形成念珠細胞，其餘Pwar-1、Ppri-1、Pdel-2則無形成念珠細胞。Pdel-1、Pdel-2、Pmic-1菌種於WA培養基的表面形成菌絲捲。以上這8株菌種的菌絲形態經初步鑑定皆為絲核菌屬的真菌。
於芭菲爾鞋蘭共生發芽方面，以這8株菌種(Pwar-1、Peme-1、Ppri-1、Phen-1、Phel-1、Pdel-1、Pdel-2及Pmic-1)、3種接種塊(直徑0.5、1.0、1.5 cm圓盤)、3種光週期(0/24 h、16/8 h、24/0 h L/D )、4種接種時間(播種前一週接種、播種同時接種、播種後1與2週後接種)及京都培養基內蔗糖濃度(5g l-1、10 g l-1、15 g l-1 、20 g l-1、25 g l-1、30 g l-1)測試對芭菲爾鞋蘭種子發芽率之影響。不論是原生種或是交配種，播種於OMA培養基並接種蘭菌的芭菲爾鞋蘭種子發芽率顯著高於播種於京都培養基的發芽率，而播種於京都培養基內部分種子發育較快，可是發芽不整齊，而播種於OMA培養基的種子之發芽率雖高，卻只發育至stage 2。接種塊的大小及接種的時間對芭菲爾鞋蘭種子發芽無顯著性影響，但接種時間愈晚發芽率愈低。Paph. delenatii播種於含10 g l-1蔗糖的京都培養基並接種Pdel-2菌種之處理有97％發芽率，其中有78％的種子發育至stage 4。
於芭菲爾鞋蘭出瓶苗接種蘭菌方面，先以8株菌種測試是否對綠豆及甘藍有致病性，再探討8株菌種(Pwar-1、Peme-1、Ppri-1、Phen-1、Phel-1、Pdel-1、Pdel-2及Pmic-1)與接種次數測試對芭菲爾鞋蘭出瓶苗生長的影響。Ppri-1菌種對綠豆有3.7％的罹病率，Ppri-1、Phen-1、Phel-1對甘藍有3.7％的致病率，Peme-1菌種對甘藍有7.4％罹病率，其他菌種對綠豆與甘藍無致病性。接種次數對Paph. delenatii fma. album的株高、株寬、葉寬、鮮重及乾重無顯著的影響，但出瓶後接種一次有較高的葉數及根數。接種8菌種中以Pwar-1、Ppri-1對Paph. delenatii fma. album的根數與鮮重均有促進的效果，Ppri-1對Paph. Maudiae Type的葉寬、根數、鮮重與乾重也有促進的效果，且葉色較濃綠，其餘的菌種對Paph. delenatii fma. album與Paph. Maudiae Type的生長則與不接種的植株無顯著性差異。
供試的8株菌種培養於黑暗環境的菌絲生長速度較培養於照光環境快速，顯示照光會抑制菌絲的生長。Pdel-2菌種最適培養條件為：25℃至30℃的環境與pH值為5.0的液體培養基。以四種液體培養基培養Pdel-2菌種，其中以V8果汁培養基的菌絲乾物量最高，其次為PDB、CM培養基，1/2 PDB的菌絲乾物量最低。震盪培養與靜置培養4週後菌絲乾物重呈明顯差異，震盪培養的菌絲乾物重高於靜置培養的菌絲乾物重。

In this study, 8 mycorrhizal Rhizoctonia-like fungi strains were isolated from roots of 7 different species Paphiopedilum, Pwar-1, Peme-1, Ppri-1, Phen-1, Phel-1, Pdel-1, Pdel-2 and Pmic-1. Pwar-1 isolate is multinucleate and the others isolates are binucleate by fluorescence light microscopy after stained with acridine orange. Monilioid cell formed in WA media by Peme-1, Phen-1, Phel-1, Pdel-1 and Pmic-1 isolates, other Pwar-1, Ppri-1 and Pdel-2 isolates have not been observed monilioid cell formed in WA media. Pdel-1, Pdel-2 and Pmic-1 isolates occurr hyphal coils on surface of WA media. Taxonomic studies revealed that these isolates belong to the genera Rhizoctonia.
The effects of 8 mycorrhizal fungal (Pwar-1, Peme-1, Ppri-1,Phen-1, Phel-1, Pdel-1, Pdel-2 and Pmic-1), inoculum block size of Pdel-2 (diameter 0.5, 1.0, 1.5 cm disc), three photoperiod treatments(0/24 h, 16/8 h, 24/0 h L/D), timing of inoculation(Before sowing seeds a week inoculation, sowing seeds also inoculation, inoculation was carried out 1, 2 weeks after sowed seeds), sucrose concentration on Hyponex medium (5 g l-1, 10 g l-1, 15 g l-1 , 20 g l-1, 25 g l-1, 30 g l-1) on symbiotic germination of Paphiopedilum. No matter what kind of species, seeds germination percentage of Paphiopedilum cultured on the OMA medium with Rhizoctonia-like fungi is higher than sowed seeds to the Hyponex medium, and sows seeds to the Hyponex medium seed partial seeds growth quicker but germination not uniform. Although germination hight on OMA medium, protocorm actually only develop to stage2. The inoculum block size and timing of inoculation were not significantly different within symbiotic germination of Paphiopedilum, but inoculation time later germination percentage is lower. The Hyponex medium contains 10 g l-1 sucrose with Pdel-2 isolate have 97% germination percentage, including 78% seed growth to stage 4.
In terms of Paphiopedilum inoculated with Rhizoctonia fungi, test first whether there is pathogenic strength to mung bean and cabbage with 8 mycorrhizal fungi, then the effects of 8 mycorrhizal fungi (Pwar-1, Peme-1, Ppri-1, Phen-1, Phel-1, Pdel-1, Pdel-2 and Pmic-1) and inoculate with the number of times on growth and development of Paphiopedilum. Results of the pathogenicity test showed that of Ppri-1 isolate has 3.7％ disease rate to the mung bean seedling. Ppri-1, Phen-1 and Phel-1 isolates have 3.7％ disease rate to the cabbage seedling and Peme-1 isolate has 7.4％ disease rate. Other isolates were no pathogenicity for mung bean and cabbage. Inoculate with the number of times not significantly different with plant height, plant width, width of leaf, fresh weight and dry weight of Paph. delenatii fma. album, and plantlets of inoculate once ex vitro have better number of leaves and roots. Paph. delenatii fma. album inoculated with Pwar-1 and Ppri-1 isolates have result promoted number of roots and fresh weight. Paph. Maudiae Type inoculated with Ppri-1 isolate has higher width of leaf, number of roots, fresh weight, dry weight and the color of leaf is relatively thick and green. Between other isolates and no inoculum were not sighificantly different with growth of Paph. delenatii fma. album and Paph. Maudiae Type.
The growth speed of these mycorrhizal fungi culture in darkness faster than culture in light condition, revealed the light inhibits the growth of the hypha. The optimal condition for growth of Pdel-2 was 25℃ to 30℃, pH 5.0 of liquid media. The Pdel-2 isolate with 4 kinds of liquid culture medium, dry weight of hypha of V8 juice liquid medium is the heaviest, secondly it is PDB, CM liquid medium, dry weight of Pdel-2 isolate hypha of 1/2 PDB is the lightest. Dry weight of Pdel-2 isolate with shaken and static cultures after 4 weeks show significantly different, dry weight of shaken cultures is higher than static cultures.

目次
壹、中文摘要……………………………………………………………i
貳、英文摘要……………………………………………………………ii
參、前言…………………………………………………………………1
肆、前人研究……………………………………………………………2
一、芭菲爾鞋蘭
(一)分類與分布………………………………………………………2
(二)產業現況…………………………………………………………4
(三)栽培與管理………………………………………………………4
(四)繁殖方法…………………………………………………………6
二、蘭菌與蘭科植物之共生關係
(一)蘭菌之定義與鑑定………………………………………………8
(二)蘭菌之分類………………………………………………………8
(三)蘭菌的感染過程及其模式………………………………………10
(四)影響蘭菌與蘭科植物建立共生關係之因子……………………11
(五)蘭菌對蘭科植物生長發育之影響………………………………13
(六)蘭菌對蘭科植物生理之影響……………………………………14
伍、材料與方法
一、蘭菌之分離與鑑定 ………………………………………………17
(一)根段來源…………………………………………………………17
(二)分離菌種前之鏡檢………………………………………………17
(三)菌種分離與純化…………………………………………………17
(四)菌種形態與細胞核數之觀察……………………………………17
(五)菌種保存………………………………………………………18
二、接種蘭菌對芭菲爾鞋蘭種子發芽之影響
(一)植物材料來源……………………………………………………18
(二)培養基之製備……………………………………………………18
(三)培養環境條件與發芽率之計數…………………………………18
(四)試驗方法
1.不同菌種對芭菲爾鞋種子發芽率之影響………………………19
2.接種塊大小對芭菲爾鞋蘭種子發芽率之影響…………………19
3.光週期對芭菲爾鞋蘭種子發芽率之影響………………………19
4.接種時間對芭菲爾鞋蘭種子發芽率之影響……………………19
5.京都培養基的蔗糖濃度對芭菲爾鞋蘭種子發芽率之影響……19
(五)原球體之組織學觀察………………………………………………19
(六)統計分析……………………………………………………………20
三、接種蘭菌對芭菲爾鞋蘭出瓶苗生長之影響及接種方式之改進
(一)蘭菌對綠豆與甘藍致病性分析…………………………………20
(二)植株材料…………………………………………………………20
(三)菌種之製備………………………………………………………20
(四)栽培介質…………………………………………………………21
(五)試驗方法
1.接種方式對芭菲爾鞋蘭接種率之影響…………………………21
2.接種蘭菌對芭菲爾鞋蘭出瓶苗生長之影響……………………21
3.蘭菌接種次數對芭菲爾鞋蘭出瓶苗生長之影響………………21
(六)根部皮層細胞於光學顯微鏡與掃描式電子顯微鏡下之觀察…21
(七)調查與統計分析…………………………………………………22
四、蘭菌生產技術試驗
(一)光照對蘭菌生長之影響…………………………………………22
(二)溫度對蘭菌生長之影響…………………………………………22
(三)養液對蘭菌生長之影響…………………………………………22
(四)培養基的pH值對蘭菌生長之影響……………………………23
(五)震盪培養對蘭菌生長之影響……………………………………23
陸、結果
一、蘭菌之分離與鑑定
(一)分離菌種前之鏡檢………………………………………………24
(二)菌種分離與純化…………………………………………………24
(三)菌種形態與細胞核數之觀察……………………………………24
二、接種蘭菌對芭菲爾鞋蘭種子發芽之影響
(一)不同菌種對芭菲爾鞋蘭種子發芽率之影響……………………25
(二)接種塊大小對芭菲爾鞋蘭種子發芽率之影響…………………25
(三)光週期對芭菲爾鞋蘭種子發芽率之影響………………………25
(四)接種時間對芭菲爾鞋蘭種子發芽率之影響……………………25
(五)京都培養基的蔗糖濃度對芭菲爾鞋蘭種子發芽率之影響……25
(六)原球體之組織學觀察……………………………………………26
三、接種蘭菌對芭菲爾鞋蘭出瓶苗生長之影響及接種方式之改進
(一)蘭菌對綠豆與甘藍致病性分析…………………………………26
(二)接種方式對芭菲爾鞋蘭接種率之影響…………………………26
(三)接種蘭菌對芭菲爾鞋蘭出瓶苗生長之影響……………………26
(四)蘭菌接種次數對芭菲爾鞋蘭出瓶苗生長之影響………………27
(五)根部皮層細胞於光學顯微鏡與掃描式電子顯微鏡下之觀察…27
四、蘭菌生產技術試驗
(一)光照對蘭菌生長之影響…………………………………………27
(二)溫度對蘭菌生長之影響…………………………………………27
(三)養液對蘭菌生長之影響…………………………………………28
(四)培養基的pH值對蘭菌生長之影響………………………………28
(五)震盪培養對蘭菌生長之影響……………………………………28
柒、討論
一、蘭菌之分離與鑑定
(一)分離菌種前之鏡檢………………………………………………29
(二)菌種形態與細胞核數之觀察……………………………………29
二、接種蘭菌對芭菲爾鞋蘭種子發芽之影響
(一)不同菌種對芭菲爾鞋蘭種子發芽率之影響……………………30
(二)接種塊大小對芭菲爾鞋蘭種子發芽率之影響…………………31
(三)光週期對芭菲爾鞋蘭發種子芽率之影響………………………31
(四)接種時間對芭菲爾鞋蘭種子發芽率之影響……………………32
(五)京都培養基的蔗糖濃度對芭菲爾鞋蘭種子發芽率之影響……32
(六)原球體之組織學觀察……………………………………………33
三、接種蘭菌對芭菲爾鞋蘭幼苗生長之影響及接種方式之改進
(一)蘭菌對綠豆與甘藍致病性分析…………………………………33
(二)接種方式對芭菲爾鞋蘭接種率之影響…………………………33
(三)接種蘭菌對芭菲爾鞋蘭出瓶苗生長之影響……………………33
(四)蘭菌接種次數對芭菲爾鞋蘭出瓶苗生長之影響………………34
(五)根部皮層細胞於光學顯微鏡與掃描式電子顯微鏡下之觀察…34
四、蘭菌生產技術試驗………………………………………………35
捌、圖表…………………………………………………………………37
玖、參考文獻 …………………………………………………………72

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