太魯閣國家公園臺灣百合內生菌群落調查

Abstract

臺灣百合(Lilium formosanum)為臺灣特有種植物，且為太魯閣國家公園內重要的觀賞作物。植物之內生微生物與其健康狀態息息相關，這些微生物可能扮演促進植物生長、拮抗病原與緩解非生物性壓力等角色。因此，了解植物內生菌相，有助於健康植株之培育。本研究目標運用高通量定序技術建構太魯閣國家公園內臺灣百合之葉片與種子內生微生物群落圖譜，並結合抗病潛能篩選的研究模式，提供培育健康臺灣百合之資訊。本研究與太魯閣國家公園管理處合作，於2020至2021年間隨機搜集公園內之臺灣百合樣本共40件。首先，針對樣本植株進行外觀檢察與常見病原菌之分子偵測，發現部分樣本雖可見病斑與微生物生長，但並非由常見病原真菌所致，判斷可能為未知病原或伺機微生物導致。隨後，本研究建立適用於臺灣百合組織的內生微生物總體DNA萃取方法，運用高通量定序技術，並搭配生物資訊學方法，進行微生物群相分析。2020年，以Illumina Miseq平台次世代定序技術（Next generation sequencing，NGS），取得該年度種子樣本中細菌16S核糖體基因的V3-V4區段序列，分析內生細菌相。結果發現，變形菌門(Proteobacteria)佔樣本中的38.8%，為優勢菌群，次要菌群厚壁菌門 (Firmicutes)僅佔1.6%。進一步註解分析OTU (operational taxonomic unit)，發現其中包含Sphingomonas paucimobilis、Proteus mirabilis、Pseudomonas fulva、Acinetobacter calcoaceticus等具促進植物生長及拮抗病原潛力等益菌。基於定序結果的分析受到葉綠體相關序列高度干擾，因此本研究後續針對臺灣百合設計封閉引子，以降低植物核酸於反應中的增幅。經過測試，最終選擇目標區段為核糖體基因V3-V4區間的引子319_chloro_F4，並於3端處加上C3 spacer修飾，完成百合封閉PCR的技術。將樣本經封閉PCR處理後，樣本內葉綠體序列的干擾下降3.2至13.5%。於2021年間，運用PacBio平台的第三代定序技術，取得該年度樣本的細菌16S核糖體基因的V1-V9區段序列，進行種子與葉片的內生菌相分析。結果發現，變形菌門(Proteobacteria) 在種子與葉片樣本中皆為主要菌群，分別佔94.6%和54.1%。於種子樣本內，其餘菌群為擬桿菌門(Bacteroidetes)和Synergistetes門。葉片樣本中之群相組成則較為豐富，其餘菌群包含12.6%放線菌門(Actinobacteria)、7.3%厚壁菌門(Firmicutes)、5.5%酸桿菌門(Acidobacteria)、及少量浮黴菌門(Planctomycetes)、疣微菌門(Verrucomicrobia)和軟壁菌門(Tenericutes)。進一步分析ASV(Amplicon sequencing variant)並比對文獻，發現18個屬共30種細菌具促進植物生長、增加作物產量、拮抗病原等潛力。比對兩年所使用不同平台進行高通量測序的結果，發現定序之序列長度會影響物種註釋結果。此外，本研究自新鮮百合組織中分離出43株內生細菌，進行培養與百合灰黴病原真菌Botrytis elliptica拮抗實驗，以篩選具有百合灰黴病生物防治潛力的分離菌株。拮抗實驗成功篩選出5個可強效拮抗該百合重要病原菌之分離株T7W-2、T5L-1、T9L-1、T10L、T12L-1。以16S rRNA與gyrB基因定序分析，鑑定T7W-2為Pseudomonas 屬細菌、T5L-1、T9L-1、T10L為Bacillus屬細菌，而T12L-1為Bacillus safensis。前人研究指出，上述2個屬內有許多菌株為植物有益細菌。本研究為臺灣百合首篇利用高通量定序與生物資訊分析進行的研究。研究成果運用高通量定序技術揭示臺灣百合微生物相的資訊，採取改善寄主序列干擾定序分析的方法，並獲取具防治潛力之有益分離株，提供日後進行百合培育研究之重要資訊。

Taiwan lily (Lilium formosanum) is endemic to Taiwan and an important ornamental crop in Taroko National Park. Previous studies have shown that endophytic microbes are closely related to the overall health of their host plants. Endophytic beneficial microbes may play roles in promoting plant growth, antagonizing pathogens, or alleviating abiotic stress. Therefore, understanding the endophytic microbial community of a plant would benefit its cultivation. The objectives of this study are to profile the leaf and seed endophytic microbial community of L. formosanum in Taroko National Park by using high-throughput sequencing technology, then combine an assay for disease resistance potential screening, and ultimately provide information for cultivation. In cooperation with the Taroko National Park Management Office, this study randomly collected 40 samples L. formosanum samples during the years 2020 to 2021. First, the appearance inspection and the PCR detection revealed that the disease symptoms and microbial growth observed on the samples were not caused by commonly known pathogenic fungi, but possibly by unknown pathogens or other opportunistic microorganisms. Subsequently, a microbial DNA extraction method for the lily tissue was established. High-throughput sequencing technology and bioinformatics were then applied to conduct microbiome analysis. In 2020, the Illumina next-generation sequencing (NGS) platform was used, and the sequences of the V3-V4 region of the bacterial 16S ribosomal gene from seed samples were analyzed. Results showed that Proteobacteria was the dominating group, accounting for 38.8% of the population, and Firmicutes was the second largest group, accounting for only 1.6% of all. Further OTU (operational taxonomic unit) analysis revealed bacterial species with plant growth promoting and pathogens antagonistic potentials, including Sphingomonas paucimobilis, Proteus mirabilis, Pseudomonas fulva, and Acinetobacter calcoaceticus. Because plant-related sequences highly interfered with the sequencing data during the analysis, this study then designed a specific blocking primer for L. formosanum to reduce the amplification of plant DNA. The primer “319_chloro_F4 with a C3 spacer modification at the 3’ end”, targeting the V3-V4 region sequence of L. formosanum, was finally selected for the blocking PCR assay. After the blocking PCR treatment, the chloroplast sequence interference decreased from 3.2 to 13.5%. For the 2021 samples, the PacBio third-generation sequencing technology (TGS) platform was used to obtain the V1-V9 region of the bacterial 16S ribosomal gene for endophytic microbiome analysis. Results showed that Proteobacteria was dominating the bacterial community in both seed and leaf samples, accounting for 94.6% and 54.1%, respectively. In the seed samples, the remaining main groups were Bacteroidetes and Synergistetes. The endophytic bacterial community in the leaf samples is relatively richer and includes 12.6% Actinobacteria, 7.3% Firmicutes, 5.5% Acidobacteria, and a small amount of Planctomycetes, Verrucomicrobia, and Tenericutes. Further analysis of the ASV (Amplicon sequencing variant) revealed that 30 species of bacteria, belonging to 18 genera, have the potential to promote plant growth, increase crop yield, and antagonize pathogens. The microbiome analysis results from the two sequencing platforms indicated that the sequencing length affects the annotation results. In addition, 43 endophytic bacterial isolates were isolated from fresh lily tissues, cultured, and screened against the pathogenic fungus Botrytis elliptica for biological control application potential. Among them, 5 isolates (T7W-2, T5L-1, T9L-1, T10L and T12L-1) have strong antagonism to B. elliptica in a dual culture assay. The sequence analysis of bacterial 16S rRNA and gyrB genes revealed that T7W-2 belongs to Pseudomonas, T5L-1, T9L-1 and T10L were Bacillus species, and T12L-1 was identified as B. safensis. Previous studies suggested that many Pseudomonas and Bacillus strains are beneficial to plants. In sum, this research was the first microbiome study of L. formosanum that used high-throughput sequencing technologies and bioinformatics. The study revealed profiled the leaf and seed microbiome of L. formosanum, conducted a blocking PCR method to reduce the interference by host sequences, and obtained bacterial isolates with biological control potential. These significant outcomes provided important information for lily cultivation.