台灣杉與林地土壤中菌根之定性與定量

Abstract

自六龜鳳岡山苗圃附近的台灣杉(Taiwaniana cryptomeriodesis)造林地根域土壤，以濕篩(wet sieving)及糖液離心法，分離出Acaulospora mellea、Scutellospora calospora和Glomus aggregatum三種較具優勢的叢枝菌根菌孢子(AMF)。應用nested PCR及菌根菌18S rRNA專一性引子(分別為GeoA1/ART4；GeoA2/Geo11)，自上述三種孢子萃取DNA，可增幅A. mellea和S. calospora近乎完整的18S rRNA及台灣杉的18S rRNA片段，將產物選殖後定序，另外以酵素切割，顯示叢枝菌根菌的18S rRNA表現具高度多型性(polymorphism)。以通用性的引子ASGf2與Geo11於nested PCR反應，可自 台灣杉根部菌根增幅734 bp G. sp.的18S rRNA。 將台灣杉和前述的三種內生菌根菌18S rRNA排齊(align)後，依據不保守區域設計個別菌種的種(specie)專一性引子(ASG1062-18S與Acau2/Scute2/Glo2)，其產物大 小約649 bp。 溫室中台灣杉幼苗接種單一菌種，約四個月後，取部分台灣杉苗木根段染色，觀察發現具有菌根感染成功。將苗木剩餘的根段以CTAB方法萃取DNA，經專一性引子進行nested PCR及選殖，並應用限制性酵素(HinfI和HaeIII)片段多型性(restriction fragment length polymorphism)篩選適當的載體進行定序。結果顯示與原始接種的菌種一致，確定上述由菌根偵測菌根菌種的方法是可行的。進一步應用到野外台灣杉的根段，證實野外共生菌種有S. calospora及G. sp.。台灣杉幼苗接種成功的A. mellea菌種，在野外偵測不到，但是可發現於林下的其他植物根部；推測在本試驗地A. mellea與台灣杉之間屬於較弱的共生關係。兩次不同時間所採的各20棵根樣本，有偵測到 菌種的樣本數量明顯第一次(92/11/24)比第二次(93/2/28)多。 利用real-time PCR配合菌種18S rRNA專一性引子(產物約649 bp)及螢光劑SYBR® Green I，可個別偵測並定量溫室接種及野外台灣杉共生菌根菌，S. calospora與G. sp.。個別帶有菌種部分SSU rRNA不同拷貝數之質體DNA的對數值(log)與定量PCR threshold循環數所繪成的標準曲線，其相關係數分別為r2=0.998及r2=0.999。應用於偵測菌根菌的感染量及野外台灣杉個別共生菌根菌的族群量變化，並以台灣杉葉部DNA為對照組，證實所測對象確實為菌根共生菌根菌。S. calospora與G. sp.兩菌種在兩次不同時間所採的樣本裡，其族群變化量明顯有差異，是否菌根菌確實具時間性動態變化，則尚需更多的採樣次數與定量數據。

Acaulospora mellea, Scutellospora calospora and Glomus aggregatum are the three dominant arbuscular mycorrhizal fungi (AMF) species isolated from rizosphere soil of Taiwania stand near Feng-Gang mountain nursery at Liou-Guei by wet sieving and sucrose centrifugation. DNAs can be extracted from individual spores from the isolated AMF and Taiwania roots. The nearly complete 18S rRNA genes of A. mellea and S. calospora can be amplified by nested PCR technigue with two 18S rRNA specific primer pairs (GeoA1/ ART4 and GeoA2/Geo11). The PCR products were cloned and sequenced. The cloned AMF 18S rRNA genes showed a polymorphism after digestion with restriction enzymes HinfI and HaeIII. A partial 18S rRNA gene (ca. 734 bp) of G. sp. was amplified from the DNA extracted from Taiwania mycorrhiza with the primer pair ASGf2 and Geo11 in a nested PCR. The sequences of 18S rRNA gene of Taiwania and three AMF described above were aligned. Based on the conserved sequences of 18S rRNA gene after aligment, the fungal species-specific primers for each species were designed. The fungal specific of each species about 649 bp DNA fragment can be PCR amplified. The colonization of AMF in the roots of Taiwania seedlings after inoculation with A. mellea and S. calospora for about four months can be verified by visualization of the trypan blue staining. DNA fragments produced by nested PCR with fungal specific primers were cloned and sequenced. The resulted sequence matched to that of the inoculated AMF. The methodology was then applied to monitor AMF within Taiwania roots from the field and the two symbiotic fungi S. calospora and G. sp. were found to be associated in. Twenty root samples were collected each time on 92/11/24 and 93/2/28. However, A. mellea can only be found in the roots of inoculated Taiwania seedlings but not the roots collected from the field. To detect and quantify the population of S. calospora and G. sp. associated with the roots of Taiwania efficiently, real-time PCR technique is setup to amplify the 18S rRNA gene (ca. 649 bp) with specific primers. Standard curves showed a linear relation (r2=0.998 and r2=0.999 each) between log values of the starting copy numbers of target sequences and real-time PCR threshold cycles. Real-time PCR can also be used to monitor dynamic variances of individual AMF communities in the field. Comparing two collects results derived from real-time PCR of S. calospora and G. sp. suggested a significant change of the colonization populations between these two collections. However, more samples from various time are required to evident whether AMF communities are actually time dynamic variances.