台灣目前栽培香菇品系與側耳屬特性之研究

Abstract

第一章 台灣目前栽培香菇品系特性之研究 香菇是廣為世界栽培的主要菇類之一，亦為台灣主要栽培的食用菇，本研究收集目前台灣地區主要栽培品系計921、.922、721、271、白種及台灣各地分離的菌株48株菌株，進行其特性分析比較。綜合菌絲生長勢與生長速度，以玉米粉為較佳的添加培養基質，而適合之碳氮比為20/1~40/1，適合pH值生長以3.5~5.6，依其菌絲生長溫度測試將收集香菇菌株分為，高溫溫度A群(Group TA)；中溫溫度B群(Group TB)，低溫溫度C群(Group TC)。液態培養測試，菌絲量培養至第20天平均乾重達228~240mg為最高，依其菌株產生原基、褐化現象、產生菌絲團等特性分析，結果將其分為液態培養A群(Group LA)、液態培養B群(Group LB) 、液態培養C群(Group LC)及液態培養D群(GroupLD)。此外，為了解菌株間品系差異，運用培養形態觀察將其台灣主要栽培菌株分為固態培養A群(GroupMA)、固態培養B群(GroupMB)、固態培養C群(GroupMC)、固態培養D群(GroupMD)、固態培養E群(GroupME)，在分群中固態培養MC群，而固態培養MA群和液態培養LA群菌落形態特性相近，在分析此群菌絲寬度與扣子體均為群1(Group1)；固態培養MB群及液態培養LC群，而固態MD與液態培養LB群菌落形態特性相近，此群菌絲寬度與扣子體均為群2(Group2)；固態培養ME與，與液態培養LD群菌落形態特性較無相似性，但是分析此群菌絲寬度與扣子體均為群3(Group3)。綜合培養條件，與固態菌落形態及液態培養菌落形態比較，A群(Group A)、F群(Group F)與J群(GroupJ)其分布於液態培養LA與LC群中，而I群因其溫度為中高溫而零星歸納在液態培養LA(GroupLA)、LB(GroupLB)、LC(GroupLC)中；而D群(GroupD)、E群(Group E)、H群(Gropu H)菌絲生長範圍較偏低溫與液態培養LD群相吻合，菌絲寬度與扣子體距離亦吻合，在固態培養呈現較多分群。爲了解菌株間歸類情形，亦運用體細胞不親和反應測試，依照其交接區的無菌絲帶寬窄，背面色素深淺及反應菌絲密度、寬度等不同，將其反應劃分為親和、微親和、間親和、不親和等四個等級，運用此篩選出21株香菇菌株，再進行更細微分子分析，並與10個國外香菇菌株與國內目前主要栽培香菇菌株比較，運用ITS4與ITS5序列分析其皆能增幅出約780 bp，而運用隨機擴增性DNA (RAPD)，篩選出10個隨機引子都可以獲得穩定的RAPD譜帶，擴增片分子量在0.18 kb-2.52 kb之間，單引子可增幅DNA片段數目在7-19個之間，運用10個引子增幅結果應用電腦軟體NTSYS-pc依UPGMA分群法進行菌株遺傳差異比較，在0.68的DNA相似係數上將其歸類為三群而台灣主要集中在一群，部分菌株與國外菌株相近，在0.91的DNA相似係數上可分為七群，亦說明台灣栽培香菇遺傳背景廣泛，ITS序列分析結果台灣栽培香菇菌株存在著相當程度的差異在ITS2序列差異較大，針對部分台灣香菇菌株在ITS序列已登錄在NCBI以供提供研究學者參考且ITS區間分析結果可用於與國外菌株進行序列比對，值得深入研究探討。

第二章 台灣目前栽培側耳屬特性之研究 側耳屬(Pleurotus spp.)為林木的主要腐生菌，對木材具有強烈的分解能力，具有研究價值。側耳屬亦為全球主要栽培的食用菌之一，極具有商業及營養價值。全世界人工栽培種有12種以上，在台灣主要的人工栽培種為P. ostreaus, P. abalones, P. cystidisus, P. eryagii等，本研究收集台灣目前主要栽培的側耳屬菌株30株，運用形態鑑定、培養條件、體細胞不親和反應，ITS序列及隨機引子S23、S24、OPA1~OPW02與本實驗室篩選的Pleurotus sp. (P6)分析比較。除此，P6菌株，在培養基質容易產生菇體，菌株子實體味鮮美且具有特殊香味，孢子印為白色，其孢子接近紡錘型，5.5~7.4±10X3.0~4.7，出菇溫度為16~25℃，在體細胞不親和反應中其與杏鮑菇(P. eryagii)對峙培養為不親和反應，但與黑美人 (P. abalonus)對峙培養呈現微親和反應，其在培養基或木屑栽培上不會與黑美人一樣形成分生孢子，但可形成擔子梗束。運用ITS1與ITS4序列分析其皆能增幅出約680 bp，而運用隨機增幅性DNA (RAPD)，篩選出4個隨機引子都可以獲得穩定的RAPD譜帶，擴增片分子量在0.25 kb-2.5 kb之間，單引子增幅DNA片段數目在2-8之間，運用RAPD結果顯示出不同側耳屬與地理分布，具有多態性，P6菌株與台灣P9菌株再RAPD分析結果較相近。針對側耳屬菌株進行真菌rDNA間隔區域限制性片段長度多態性分析，結果分為七大群，而秀珍菇與P6菌株有相近切位，此外亦區分開運用體細胞不親和反應的菌株P5與P9，結果可用於種內多樣性的研究，值得深入研究探討。此外位了解不同時期側耳屬基因的表現，篩選P6菌株，進行反轉錄聚合酵素連鎖反應，再進行cDNA之隨機引子擴增核酸多形性分析，選殖出A、E、F等基因片段，與NCBI上已登錄基因序列比較，發現其可能為此菌發育過程中重要基因，則有待更進一步的證明與研究。

Cheaper 1 Study on the characteristics of present cultural strains of Lentinula edodes in Taiwan

Lentinula edodes(Berk.) Pegler,the shiitake mushroom, is one of the most widely cultivated mushrooms worldwide. The study of the project is to investigate the characteristics of different isolates and cultivated strain 921,922,721,271 and white strain of the Lentinula edodes in Taiwn.The results indicate that the optimal medium was corn powder, the optimal C/N ratio was 20:1~40:1 and the optimal pH value was 3.5~5.6.Based on the temperature to mycelial growth, divided into high,middle,and low temperature group. In high temperature group include groupTA, in middle temperature group includes group TB, in low temperature group includes groupT C. Based on liquid cultural characteristics divided into GroupTA、TB、TC,and the optimal liquid culture medium was extract-peptone-dextrose.The optimal growth curve was twenty days and follow their characteristics of primordium, the appearance of browing and mycelium rounds. Based on the above characterization divided into GroupLA、GroupLB、GroupLC、GroupLD. Besides, in order to understand the differentence strains of L.edodes,using culture morphogenesis observation divided into GroupMA、GroupMB、GroupMC、GroupMD、GroupME. The solid culture group MA , and solid culture group MC is similar to colony morphology of liquid culture group LA . Analysis the width of mycelium and the distance of clamp connection this group is belong to group1. The solid culture group MD, liquid culture group LC, and the colony morphology character of solid culture MB group is similar to liquid culture group LD. Analysis the width of mycelium and the distance of clamp connection this group is belong to group2. The colony morphology of solid culture group ME and liquid culture group LD are not the same. Analysis the width of mycelium and the distance of clamp connection this group is belong to group3. In conclusion culture condition, comparing solid culture group and liquid culture group, we found groupA, groupF, groupJ are belong to liquid culture group LA and LC. Group I was dispersed to liquid culture group LA, LB and LC,but group D, groupE and group H are belong to LD. Analysis the width of mycelium and the distance of clamp connection are accord with before results. But the solid culture group are more complex. Becasue of the different environment affecting the morphogenesis of L.edode,somatic incompatility mating type analysis divided into compatilibity,slight compatilibity,intercompatilibity,incompatilibity etc. four level.Besides,in order to select the molecular mark to discriminate to forgein countries using DNA extraction to comparing with the strains of forgen and Taiwan strains.PCR products produced with primer ITS1 and ITS4 were visualized as a single band.The size of the PCR fragments was 720bp. Single 10-base primers were used to generate randomly amplified polymorphic DNA(RAPD) markers in the shiitake mushroom, Lentinula edodes. Ten primers produced polymorphisms in all 31 strains tested, producing 7-19 bands ranging from 0.18 to 2.52 kb.10 primers results from 27 primers RAPD profiles with amplification of 31 L.edodes isolates were select to analysis genetic similarity by UPGMA cluster analysis with computer software NTSYS-pc. We found that isolates of L. edodes have different genetic diversity. It indicated that 31 isolates of L.edodes could be divided into severn group under DNA similar coefficient of 0.68 mainly in Taiwan, some isolates is similar to foreigen countries. It indicated that 31 isolates of L.edodes could be divided into severn group under DNA similar coefficient of 0.91. It indicated that Taiwan cultivate L.edodes have rich genetic diversity.Internal transcribed spacer sequences analysis result Taiwan cultural L.edodes. and ITS2 region explose variability. Have already logged in for offering a researcher to consult in NCBI in ITS sequences to some isolates of L.edodes in Taiwan and worth further investigating.

Cheaper 2 Study on the characteristics of present cultural Pleurotus spp. in Taiwan

Pleurotus spp.is the main white rot fungi in forest.They have ability to decompose wood.There are about twelve varieties were cultivated worldwith. But in Taiwan,there are some varieties included P. ostreaus, P. abalones, P. cystidisus, P. eryngii were cultured. P6’s cap is 2~13.0cm across,convex at first,then becoming plane to broldly depressed when old. it’s surface dull, moist, smooth, glabrous, whitish to cream in colour. In the medium, it can form deerhorn hyphae and then form the fruit body.it’s spore elliiptical to subfusiform, smooth,about 5.5~7.4±10X3.0~4.7. The optimal fruiting temperature is 16~25℃. Somatic incompatility mating type analysis of that it was moer closed P. abalonus than P. eryngii. And the culture forms coremia , but do not form conidia on the medium or sawdust substrate.PCR products produced with primer ITS 1 and ITS 4 were visualized only a single band. The size of the PCR fragments was 680 bp. Single 10-base primers were used to generate randomly amplified polymorphic DNA (RAPD) markers in the P. spp. Four primers produced polymorphisms in all tested 30 strains, producing 2-8 bands ranging from 0.25 to 2.50 kb.Molecular genetic markers obtained with the RAPD assay can be used to differentiate isolates of P. spp. and have potential applications in mushroom breeding and strain improvement programs. Besides, we also analysis internal transcribed spacer of Pleurotus spp and login NCBI. The internal transcribed spacer explose variability in L. edodes and worth further investigating. We also used internal transcribed spacer- restriction fragment length polymorphism to analyze P. spp. It can distinguish P. spp. as seven groups and P.ostreatus(white) had the same DNA fragments with P6.Using ITS-RFLP analysis also could divide P9 and P5 into different group that we could not divide P9 and P5 into different group by somatic incompatility mating. To analysis genes involved in fruit body development of P6 , mRNA from three different developmental stages : vegetative mycelium, and fruit body. Twenty random PCR amplifications were performed with the cDNAs , whicg A, E, F, cDNA fragments from different developmental stage. Sequence analysis and database searches reveraled significant similarity with Ustilago maydis hyposis protein, Nurospora crassa filment H protein.