陽明山國家公園重金屬土壤細菌群落調查及臺灣二葉松菌根於該土壤對細菌群落結構與重金屬濃度之影響

Bacterial Composition in Heavy-metal Soils from Yangmingshan National Park and Effect of Pinus taiwanensis Mycorrhiza on the Soil Bacterial Community Structure and Heavy-metal Concentration

宋英平(Song Ying-Ping)

指導教授 ： 吳羽婷

國立屏東科技大學/農學院/森林系所/碩士(2019年)

https://doi.org/10.6346/NPUST201900012

摘要

本研究分為兩部分，第一部分分析陽明山國家公園境內三處不同火山土壤(冷水坑、竹子湖及磺嘴山自然保護區)之細菌群落結構，了解該特殊地質環境所孕育的土壤細菌群落結構外，進一步探討不同火山土壤的物理化學性質對土壤細菌群落結構的影響。第二部分探討臺灣二葉松(Pinus taiwanensis)幼苗接種不同外生菌根菌對陽明山含有重金屬的火山土壤復育情況，以及其對根部、原試土與根域土壤的細菌群落結構變化的影響。第一部分的研究結果顯示，陽明山地區三處不同火山土壤細菌群落結構明顯不同。竹子湖地區土壤細菌Shannon多樣性指數顯著高於冷水坑及磺嘴山自然保護區。Proteobacteria是磺嘴山自然保護區土壤中最具優勢的細菌門，Actinobacteria、Acidobacteria則在冷水坑地區、竹子湖地區土壤中具有較高的相對豐富度。pH值、有效磷、有機質、Zn、Cu、Pb、Cr都是影響三種不同火山土壤細菌群落結構的重要因子(P< 0.05)。
第二部分研究中使用的兩株外生菌根菌，一株為本研究自臺灣二葉松根部分離出之外生菌根菌Pezicula ericae K2，另一株為彩色豆馬勃(Pisolithus tinctorius) (作為正對照組)。在不同濃度重金屬固體培養基試驗中，彩色豆馬勃、Pez. ericae K2在生長後期不同濃度之間的菌落直徑幾乎無顯著差異，且仍持續生長，顯示鋅濃度0-30 ppm及銅和砷濃度0-20 ppm，皆為彩色豆馬勃及Pez. ericae K2可耐受之範圍。菌落直徑的生長狀況顯示，Pez. ericae K2對重金屬鋅、銅、砷之耐受性的表現皆比彩色豆馬勃佳。將兩種外生菌根菌分別接種於臺灣二葉松苗木，並移植試驗用土壤，6個月後分析土壤化學性質變化以及植體重金屬累積。結果顯示，接種Pez. ericae K2與彩色豆馬勃處理的苗木生長量顯著高於未接種對照組。接種外生菌根菌之處理能顯著提升土壤K+、Na+、Ca2+、Mg2+含量、pH值，而土壤重金屬Zn2+、Cu2+、As3-含量則顯著降低。在不同處理下根部重金屬累積量皆比地上部高，以總累積量檢視，接種Pez. ericae K2處理可以吸收最多之重金屬，顯示外生菌根之吸收效應。接種後的根域土與根尖之細菌群落結構明顯有差異，根域土優勢菌群主要為酸桿菌門 (Acidobacteria)、變形菌門 (Proteobacteria)、綠彎菌門 (Chloroflexi)，而不同處理下之根域土多樣性指數無顯著差異。根域土細菌群落結構的變化受土壤pH值、Mg2+、Ca2+、Na+、K+、Zn2+、Cu2+、As3-、全氮量等環境因子顯著影響(P< 0.05)。外生菌根根尖與未接種根尖細菌群落結構具顯著差異，且外生菌根根尖物種豐富度與多樣性指數顯著高於未接種根尖。優勢菌群主要有放線菌目(Actinomycetales)、伯克霍氏菌目(Burkholderiales)、根瘤菌目(Rhizobiales)，其中Burkholderiales、Rhizobiales是與植物共生重要的固氮細菌。根尖細菌群落結構與重金屬Zn2+、Cu2+及As3-皆有顯著相關性。Frankia sp. 只在接種Pez. ericae K2根尖中被檢測到，這可能是Pez. ericae K2在降低土壤重金屬含量的過程中具有重要交互作用細菌群。綜合上述，Pez. ericae K2在本研究中顯示具有較佳的重金屬吸附能力，且有正向交互作用的細菌群。這些結果反映了外生菌根菌、土壤化學性質、重金屬、細菌群落結構以及宿主植物之間潛在複雜的交互關係。將來以外生菌根復育重金屬土壤時，建議可以同時接種外生菌根菌與細菌，使重金屬土壤復育更有效率。

關鍵字

外生菌根菌 ； 重金屬 ； 細菌群落結構 ； 次世代定序 ； 交互作用 ； 復育

並列摘要

The study consists of two parts. The first part was to analyze the bacterial community structure of three different volcanic soils (Lengshueikeng, Jhuzihhu and Mt. Huangzui Ecological Protected area) from Yangmingshan National Park (YNP), and to investigate the effects of soil physicochemical properties on soil bacterial community structure. The second part was to explore the capacity of Pinus taiwanensis inoculated with ectomycorrhizal fungi (ECMF) to reduce the heavy metals in the soil from YSP and its effects on the bacterial community structure in the root tip of the host plant and rhizospheres soil. The results of the first part showed that the bacterial community structure of three different volcanic soils in YNP significantly differed. Shannon diversity index of the Jhuzihhu area soil was significantly greater than both Lengshueikeng area and Mt. Huangzui Ecological Protected area. Proteobacteria was the most dominant bacterial phylum in the soil of Mt. Huangzui Ecological Protected area while Actinobacteria and Acidobacteria were the most dominat phyla in Lengshueikeng and Jhuzihhu, respectively. The environmental explanatory including pH, available phosphorus, total nitrogen, organic matter, Zn2+, Cu2+, Pb2+, and Cr3+ explained the soil bacterial community structure in the three different volcanic soils with P value less than 0.05.
In the second part, two ECMF were utilized in this study including Pezicula ericae K2 was isolated from the roots of the Pinus taiwanensis pure stand at Hehuan Mountain, and the other one is Pisolithus tinctorius (as a positive control). The experiment of fungal growth in solid agar of different heavy-metal concentration showed no significant difference in the diameter of colonies of both Pis. tinctorius and Pez. ericae K2 at the late stage, and colonies were continued to extend. Therefore Pis. tinctorius and Pez. ericae K2 were tolerant of Zn (0-30 ppm), Cu and As (0-20 ppm). However, the status of colonies showed that the Pez. ericae K2 had greater tolerant capacity to heavy-metal than Pis. tinctorius. In further, Pin. taiwanensis seedlings were successfully confirmed to be inoculated with the two ECMF. The ECM Pin. taiwanensis seedlings had been transplanted in heavy-metal soil for 6 months for the subsequent experiment. After harvesting, plant biomass (dry weight) of the treatment with inoculation of Pis. tinctorius and Pez. ericae K2 was significantly higher than that of the treatment without inoculation of ECMF. Moreover, in the treatment with ECMF, soil K+, Na+, Ca2+, Mg2+ and pH were significantly increased, while soil heavy metals including Zn2+, Cu2+, and As3- were significantly reduced. The accumulation of heavy metals in the roots was generally higher than the aboveground (stem and leaves) across all treatments. According to the total accumulation of heavy metals, the treatment with inoculation of Pez. ericae K2 had absorbed the highest amount of heavy metals, indicating the absorption capacity of ectomycorrhizal, specifically Pez. ericae K2. The bacterial community structure of rhizosphere soil and root tip significantly differed across all treatments. The soil bacterial assemblage was dominated by Acidobacteria, Proteobacteria, and Chloroflexi. The Shannon diversity index of rhizosphere soil has no significant difference across all treatments. The variation of bacterial community structure in the rhizosphere soil was affected by the environmental factors including soil pH , Mg2+, Ca2+, Na+, K+, Zn2+, Cu2+, As3-(P< 0.05). The bacterial community structures of ectomycorrhizal root tips and non-mycorrhizal root tips were significantly different, and the species richness and diversity index of ectomycorrhizal root tips were significantly higher than non-mycorrhizal root tips. In addition, Actinomycetales, Burkholderiales, Rhizobiales were the most three dominant orders, among which Burkholderiales and Rhizobiales are important nitrogen-fixing bacteria associated with plants. The variation of bacterial community structure of root tips was significantly droved by the heavy-metals including Zn2+, Cu2+ and As3-. Frankia sp. was only detected in the root tip of Pez. ericae K2 mycorrhiza, which might play an important role with Pez. ericae K2 in the reduction of soil heavy-metals. In conclusion, Pez. ericae K2 were able to significantly reduce the heavy metals and showed a better capability than Pis. tinctorius to absorb soil heavy metals in this study, and had positive interaction with the associated bacteria. In other words, the results indicated that ECMF, soil chemical properties, heavy-metals, bacterial community structure, and host plants had potential interactions. In the future, remediation of soil heavy metals by ectomycorrhizal fungi, we recommend to inoculate both ECMF and bacteria to achieve more effective remediation work.

並列關鍵字

ectomycorrhiza fungi ； heavy-metal ； bacterial community structure ； next-generation sequencing ； interaction ； remediation

參考文獻

王孟宇 (2013) 生物炭對叢枝菌根菌產孢量和宿主植物生長之影響。國立屏東科技大學森林系所碩士學位論文，143頁。

王義仲、許立達、林敏宜、林志欽 (2003) 陽明山國家公園之長期生態研究-植被變遷與演替調查。內政部營建署陽明山國家公園管理處委託研究報告，96頁。

余炳盛、劉金龍、呂祺竹 (2004) 陽明山國家公園土壤重金屬含量調查及其地質意義之探討。內政部營建署陽明山國家公園管理處委託研究報告，154頁。

呂斯文、張喜寧 (1998) 菌根菌與土壤微生物間之交互作用。科學農業 46(5,6): 217-225。

林瑞進、李明仁、王也珍 (2010) 台灣杜鵑與內生菌 Cryptosporiopsis sp. 共生形成杜鵑類菌根。中華林學季刊 43(1): 171-179。