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黏帚黴菌Gliocladium virens WJGV2、TLGV22之生物特性及其在病害防治應用厚膜孢子製劑之量產
朱盛祺
本研究之目的在篩選本土性優異黏帚黴菌菌株，且以傳統攪拌式發酵槽建立厚膜孢子製劑之液體發酵量產技術，以供病害防治之應用，並期作為此類益生性微生物資源開發應用之參考。經由厚膜孢子產孢能力與促進甘藍幼苗植株生長測試，對三個木黴菌屬菌株與四個黏帚霉黴菌屬菌株進行篩選，最後選用分別由大里、烏日水稻根圈土壤分離獲得，生長產孢特性與促進植株生長俱優異之黏帚黴菌 TLGV22 與 WJGV2兩菌株作為日後試驗之供試菌株，再經於PSA平板行對峙培養測試，證實對於立枯絲核菌Rhizoctonia solani AG1、AG4及腐霉病菌Pythium aphanidermatum均有不錯的拮抗能力；酵素活性測試並發現，兩菌株對澱粉、幾丁質、蛋白質及纖維素等天然基質均有很好的分解能力，而果膠分解酵素（PG與PTE）及脂質分解酵素活性則均未檢測出；在PSA平板上，兩菌株菌絲體皆呈無色且分支繁複，分生孢子梗直立或略微彎曲，瓶狀孢子梗呈圓錐狀，著生於分生孢子梗頂端，分生孢子均為綠色或黃綠色，橢圓型至卵圓型，單孢單室具有黏性，常在瓶狀梗上集結成一團；上述產孢型態特性檢視結果，顯示此二菌株與Domsh氏、Harman氏與Kubicek氏等所描述之Gliocladium virens特性一致。進一步利用PCR增幅技術，由兩菌株個別選殖其核糖體DNA內轉錄區間(Internal transcribed spacer，ITS)，所獲得包含ITS全長的652 bp片段 ，分別將其解序完成後與NCBI之GenBank既有資料比對，證實與已登錄之9個Gliocladium virens (syn. Trichoderma virens) 相似度均高達99-100%，綜合上述產孢型態與核糖核酸ITS區序列分析結果，將兩菌株鑑定為G. virens。於產孢應用上，TLGV22 與 WJGV2兩菌株已經證實同樣具有優異的厚膜孢子產孢特性，利用本研究正建立改進中的培養技術，於搖瓶液態培養7天，厚膜孢子產量均可達到每毫升1.5X108個以上，所產出之成熟厚膜孢子大小約為9.18-10.8μm，此一厚膜孢子懸浮液經4℃低溫儲藏六個月，孢子的存活數仍有每毫升107個以上，但在室溫下儲藏則其安定性並不如理想；擴大至50 L發酵槽量產測試厚膜孢子，發現添加碳素源含量較高的M生長因子、導入一厚膜孢子轉化促進因子(CSF)與CSF用量之最適化均有助於促進厚膜孢子的分化和提升產量，目前培養5天產量已可達108/ml以上。且此一技術平台於木黴菌與黏帚黴菌厚膜孢子量產之泛用性與750 L發酵槽擴大量產之應用性也已獲證實。所生產的厚膜孢子製劑可應用於葉部噴灑、土壤澆灌、種子浸種與被覆等處理，在溫室盆缽試驗中，利用WJGV2與TLGV22菌株所研製之厚膜孢子製劑經溫室試驗，已證實對於甘藍和水稻幼苗均有顯著的生長促進效果，其中以WJGV2菌株之厚膜孢子培養液經50倍稀釋後用於甘藍、水稻種子種植前浸種處理，其中甘藍經種植10週後調查，處理組較以水處理的對照組鮮重增加約115.2%，另外水稻則經種植2週後調查，其處理組株高增加約58.9%，處理組與對照組間差異均達顯著水準；另外在病害防治應用上，利用TLGV22與 WJGV2菌株之厚膜孢子培養液，以浸種或澆灌的方式處理甘藍防治Rhizoctonia solani AG4之立枯病，效果均顯著，對照組的存活率為13.87%，WJGV2浸種50倍稀釋處理組約較對照組提高38.91%達最顯著，而TLGV22澆灌2000倍稀釋處理組約較對照組提高33.35%；以 105 chlamydospores/ml濃度澆灌處理於人工接種Pythium aphanidermatum的甘藍幼苗，其對照組的存活率為70%，TLGV22處理組約較對照組提高21.2%，WJGV2更提高30%，此些防治效果顯示，利用兩供試黏帚黴菌菌株所研製之厚膜孢子製劑在此兩大重要土傳性真菌病害之防治，確具顯著效果。另外在水稻紋枯病防治效果評估方面，利用製劑滴施處理能有效降低田間紋枯病之發病率，較對照組降低7.85%，或減緩已感病植株病勢之蔓延而使產量損失減少，較對照組產量平均濕重增加10.9%、乾重增加12.9%、稔實率增加9.8%，證實WJGV2厚膜孢子液劑確實具有防治水稻紋枯病之應用潛力。

Biological characteristics of Gliocladium virens WJGV2 and TLGV22 and the mass production of chlamydospore formulation for disease control
Sheng-Chi Chu
The investigation was aimed to establish technique platform for exploring the Gliocladium/Trichoderma microbial resources for plant disease control application. A primed goal attempted was to produce the robustious chlamydospore biomass, rather than the conidial formulation, by a traditional stirrer tank fermentor. A total of seven antagonistic Gliocladium/Trichoderma strains with superior growth and conidiation characteristics were screened for the competence of chlamydospore production and plant growth promotion. Gliocladium strains WJGV2 and TLGV22 were among them the best in promoting the growth of cabbage seedlings and in chlamydospore productivity. The two strains are both rhizosphere isolates from rice. They both showed superior antagonisity on Rhizoctonia solani AG1, AG4, and Pythium aphanidermatum. They also expressed great hydrolytic activities on macro biomolecules including starch, protein, chitin and cellulose, whereas not on pectin or phospholipid. The morphological characteristics of mycelial growth and conidiation indicated the 2 strains accorded well the species characteristics of Gliocladium virens described by Domsh, Harman and Kubicek. The internal transcribed spacer (ITS) region rDNA of the two strains that include ITS1, 5.8S rDNA, and ITS2, was cloned and sequenced. The result obtained from sequence analysis indicated the cloned sequences from these 2 test strains were 652 bp in length which shared 99-100 % identity as compared to that of the 9 existing Gliocladium virens (syn. Trichoderma virens ) registed in GenBank of NCBI. The high identity of full length rDNA ITS region comparing to that known in GenBank further strengthen the view that to two test strains are members of G. virens.
For mass production of chlamydospore formulation, improvement of cultured medium constituents was attempted. In a flask shaking culture system, the amendment of carbohydrate-enriched growth factor appeared to increase the chlamydospore production greatly; whereas the amendment of N-enriched growth factors Y & F, on the contrary, greatly reduced it. The yield of chlamydospore in flask culture system reached 1.5X108 /ml 7 days after inoculation, and size of the produced spores ranged from 9.2 to 10.8 μm in diameter. The viable chlamydospores in the culture dropped to 107 /ml level gradually after 6 months storage at 4℃, indicating the need of protective measurement in the formulation process. In 50 L stirrer tank fermentor system, the improved broth medium developed in flask culture system was found repeatable; the yield of chlamydospore reached 108 level as expected. However, the production protocol takes 8-9 days operation. A chlamydospore formation stimulation factor (CSF) was found useful in initiating the sporulation process. The timing and dosage of CSF application had been optimized. With the introduction of 0.25 units CSF at the starting point of the culture process, the chlamydospore yield reached the expected 108 /ml level in 5 days. The improved protocol established significantly shortened the production time and greatly improved the spore uniformity. The usefulness of the established technique platform have been testified in 50 L tank system with the use of 2 strains of Trichoderma spp. and 2 strains of Gliocladium spp., and as well with test strain WJGV2 in a 750L tank fermentor.
The liquid formulation produced can be applied by foliar spray, soil drenching and seed soaking/coating. With the application by soil drenching and/or seed soaking of WJGV2 and TLGV22 each at appropriate concentration, substantial growth promotion on cabbage and rice has been demonstrated. In the case of cabbage, the seed soaking with WJGV2 formulation at 50X dilution led to 115.2 % increase in fresh weight 10 weeks after treatment. Whereas for rice, the same treatment led to 58.9 % increase in plant height 2 weeks after application. In a greenhouse system, the application of both strains was also shown effective in controlling Rhizoctonia solani AG4 and Pythium aphanidermatum seedling damping off infections on cabbage. For cabbage seeded on substrates artificially inoculated with R. solani AG4, seed soaking with WJGV2 at 50X dilution led to a 38.9 % increase in survival; likewise, soil drenching with TLGV22 at 2000X in dilution led to a 33.4 % survival increase. As for seedling damping off caused by P. aphanidermatum, soil drenching by WJGV2 and TLGV22 led to 30 % and 21.2 %, increase respectively, in percent survival. The chlamydospore formulation developed from the 2 strains thus appeared to be effective as biofungicide in the control of these 2 important soil-borne fungal pathogens. Moreover, the possible application of these 2 attempted biofungicides in the control of sheath blight on rice (R. solani AG1) was also investigated. The test formulation was applied by dripping as liquid formulation and by spraying as floatable granule formulation. The applied biofungicide treatment significantly reduced the sheath blight infection and resulted in substantial yield increases. Their potential for the control of rice sheath blight appeared to be promising and worth great attention.

目錄
壹、前言----------------------------------------------------1
貳、前人研究------------------------------------------------3
參、材料與方法---------------------------------------------14
一、供試菌株及植物材料-----------------------------------14
二、供試藥品材料來源-------------------------------------14
三、供試菌株之分離與篩選---------------------------------15
(一) 厚膜孢子產能比較--------------------------------15
(二) 厚膜孢子發酵液對甘藍幼苗生長之影響--------------16
四、對重要土傳病原之拮抗活性測試-------------------------16
五、生物巨分子分解特性測試---------------------------------17
六、WJGV2、TLGV22兩供試菌株之生物特性與種之鑑定------------19
七、rDNA ITS區間序列分子特性之比對-------------------------19
(一) 供試菌株總量DNA之抽取---------------------------19
(二) 含5.8S rDNA ITS 區間序列之增幅------------------20
(三) rDNA增幅片段之選殖解序------------------------- 20
八、五種不同培養配方對厚膜孢子形成之影響-------------------22
九、不同濃度菌絲接種源對厚膜孢子形成之影響-----------------22
十、厚膜孢子擴大量產技術之改進-----------------------------23
(一) 代號Y、M、F三種生長因子之添加對供試菌株WJGV2厚膜孢
子形成之影響------------------------------------23
(二) WJGV2與TLGV22供試菌株於生長最適化培養基厚膜孢子產量
比較--------------------------------------------23
(三) 不同時間導入厚膜孢子轉化促進因子(CSF)對厚膜孢子形 成之影響--------------------------------------- 23
(四) CSF用量對厚膜孢子形成之影響---------------------24
(五) CSF最佳化流程於黏帚黴菌/木黴菌厚膜孢子量產之泛用性-- -----------------------------------------------24
(六) CSF最佳化流程擴大到750 L發酵槽之應用性----------24
十一、厚膜孢子之儲藏安定性-----------------------------------------------------------25
十二、厚膜孢子製劑對甘藍與水稻幼苗生長之影響------------------------------- 25
(一) 浸種處理對甘藍幼苗生長之影響-------------------------------------------25
(二) 澆灌處理對甘藍幼苗生長之影響-------------------------------------------25
(三) 被覆處理對甘藍幼苗生長之影響-------------------------------------------26
(四) 浸種處理對水稻幼苗生長之影響-------------------------------------------26
十三、對甘藍立枯絲核菌Rhizoctonia solani AG4幼苗立枯病之防治效果-------26
(一) 立枯絲核菌帶菌泥碳土之製作--------------------------------------------- 26
(二) 浸種與澆灌處理對幼苗立枯病之防治效果------------------------------ 27
(三) 澆灌、被覆及浸種處理對幼苗立枯病之防治效果----------------------27
十四、對甘藍腐霉病菌Pythium aphanidermatum幼苗猝倒病之防治效果------27
(一) 腐霉病菌帶菌泥碳土之製作-------------------------------------------------27
(二) 澆灌處理對甘藍幼苗猝倒病之防治效果--------------------------------- 28
十五、WJGV2厚膜孢子液劑、漂浮性粒劑製作及其在水稻紋枯病之防治
應用---------------------------------------------------------------------------------- 28
(一) 厚膜孢子液劑與漂浮性粒劑之製作--------------------------------------- 28
(二) 液劑與漂浮性粒劑施用對台農71號(TNG71) 水稻紋枯病之防
治效果------------------------------------------------------------------------- 28
(三) 液劑滴施對台梗8號(TK8)水稻紋枯病之防治效果-------------------- 29
肆、結果----------------------------------------------------------------------------------------30
一、 供試菌株之分離與篩選------------------------------------------------------------- 30
二、WJGV2、TLGV22兩供試菌株生物特性-----------------------------------------30
(一) 對重要土傳病原之拮抗活性-------------------------------------------------31
(二) 生物巨分子分解特性----------------------------------------------------------31
(三) 產孢型態特性與種之鑑定----------------------------------------------------31
三、 rDNA ITS區間序列分子特性之比對--------------------------------------------32
四、五種不同培養配方對厚膜孢子形成之影響------------------------------------33
五、不同濃度菌絲接種源對厚膜孢子形成之影響---------------------------------33
六、厚膜孢子擴大量產技術之改進----------------------------------------------------33
(一) 代號Y、M、F三種生長因子之添加對供試菌株WJGV2厚膜孢
子形成之影響------------------------------------------------------------------- 33
(二) WJGV2與TLGV22供試菌株於生長最適化培養基厚膜孢子產量
比較------------------------------------------------------------------------------- 34
(三) 不同時間導入厚膜孢子轉化促進因子(CSF)對WJGV2菌株厚膜
孢子形成之影響---------------------------------------------------------------- 34
(四) CSF用量對厚膜孢子形成之影響--------------------------------------------35
(五) CSF最佳化流程於黏帚黴菌/木黴菌厚膜孢子量產之泛用性----------36
(六) CSF最佳化流程擴大到750 L發酵槽之應用性--------------------------36
七、厚膜孢子之儲藏安定性------------------------------------------------------------37
八、厚膜孢子製劑對甘藍與水稻幼苗生長之影響---------------------------------37
(一) 浸種處理與澆灌處理對甘藍幼苗生長之影響----------------------------37
(二) 被覆處理對甘藍幼苗生長之影響-------------------------------------------38
(三) 浸種處理對水稻幼苗生長之影響-------------------------------------------38
九、厚膜孢子製劑對甘藍立枯絲核菌Rhizoctonia solani AG4幼苗立枯病
之防治效果---------------------------------------------------------------------------38
(一) 以WJGV2與TLGV22菌株行浸種與澆灌處理之防治效果------------38
(二) 以WJGV2菌株行澆灌、種子被覆及浸種處理之防治效果-----------39
十、厚膜孢子製劑對甘藍腐霉病菌Pythium aphanidermatum幼苗猝倒病
之防治效果-------------------------------------------------------------------------- 39
十一、WJGV2厚膜孢子製劑滴施與漂浮性粒劑噴佈對水稻紋枯病之防治
效果-田間試驗---------------------------------------------------------------------- 39
(一) 液劑滴灌與漂浮性粒劑噴佈對台農71號(TNG71) 水稻紋枯病
之防治效果---------------------------------------------------------------------- 40
(二) 液劑滴灌對台梗8號(TK8)水稻紋枯病之防治效果---------------------40
伍、討論-----------------------------------------------------------------------------------------41
陸、中文摘要-----------------------------------------------------------------------------------49
柒、英文摘要-----------------------------------------------------------------------------------51
捌、參考文獻-----------------------------------------------------------------------------------55
玖、圖表說明-----------------------------------------------------------------------------------62
拾、附錄-----------------------------------------------------------------------------------------89

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