探討螢光假單胞菌 K1-3-1 誘導草莓抗炭疽病之防禦反應與標誌基因

每年四到九月為臺灣草莓栽培重要的育苗期，日益加劇的高溫，使育苗期的病害發生更加嚴峻，造成嚴重的經濟損失。其中，又以炭疽病菌 (Colletotrichum gloeosporioides complex) 所引起的草莓炭疽病最為嚴重。相較於農民習慣的「老株育苗法」，以不帶特定病原的組織培養苗馴化後作為母株育苗，能夠降低炭疽病菌藉由種苗帶原進入本田，造成本田期初期大面積幼苗死亡的風險。組織培養苗雖是很好的育苗母株選擇，但較走蔓苗育成之植株脆弱，若施用促進植物生長微生物 (Plant growth promoting microbes, PGPMs) 進行生物健化 (Biohardening)，應能提升草莓組織培養苗馴化時的生長勢與對抗逆境之能力。先前自健康草莓根系分離到的螢光假單胞菌 (Pseudomonas fluorescens) K1-3-1 能提高馴化後草莓組織培養苗對炭疽病的抗性，成為防治草莓炭疽病的一道曙光。本研究以轉錄體分析 (Transcriptomic analysis) 探討 P. fluorescens K1-3-1 提高草莓抗炭疽病菌能力的可能機制，並試圖找尋 P. fluorescens K1-3-1 處理後之關鍵標誌基因 (Marker genes)，作為日後檢測指標。結果發現，經 P. fluorescens K1-3-1 處理的草莓與對照組相比有 44 個差異表現基因， 16 個表現差異較為顯著的基因當中，有 5 個基因會專一性地受到 P. fluorescens K1-3-1 誘導表現，因此推論其具有成為標誌基因之潛力。此外，經 P. fluorescens K1-3-1 處理二天的植株，其防禦反應基因會被誘導表現；而經 P. fluorescens K1-3-1 處理後再馴化七天的草莓，染色質重塑 (Chromatin remodeling) 相關基因會被誘導表現。前人研究可知 PGPMs 可透過染色質重塑引發植物的促發效應 (Priming effect)，使植物再次遇到逆境時能更快速或更強烈地誘導防禦反應。檢測草莓感染炭疽病後防禦基因的表現情形發現，相較於未經 P. fluorescens K1-3-1 處理之草莓，經 P. fluorescens K1-3-1 處理的植株，於炭疽病菌感染 72 小時內，防禦相關基因如： NPR1、WRKY70、PR1 與 PR5，會更為快速、強烈且長時間地被誘導。此結果可推測 P. fluorescens K1-3-1 會經由促發效應使草莓在感染炭疽病菌時，更快速且強烈地誘導防禦基因之表現，以提高草莓抗炭疽病之能力。由本研究結果得知 P. fluorescens K1-3-1 可能是透過染色質重塑的方式引起草莓的促發效應，進而提升草莓對抗炭疽病之能力，未來可更深入地探討相關機制。

關鍵字

草莓炭疽病；螢光假單胞菌；生物健化標誌基因；染色質重塑；促發效應

並列摘要

April to September is the vital period for strawberry seedling production in Taiwan. However, increasing high temperature worsens the disease problem during strawberry seedling production, leading to huge economic losses. Strawberry anthracnose caused by the Colletotrichum gloeosporioides species complex is the most severe disease. Compared to conventional farming, using specific-pathogens-free (SPF) seedlings as mother plants in the strawberry nurseries could reduce the risk of transmission of anthracnose by seedlings transportation. Micropropagated strawberry seedlings are good choice as mother plants, but they are weaker than those developed from runners. Fortunately, biohardening of micropropagated strawberry seedlings with plant growth promoting microbes (PGPMs) could enhance the growth potential and ability to overcome stress during acclimatization. In a previous study, Pseudomonas fluorescens K1-3-1 was isolated from roots of a healthy strawberry plant, which could enhance strawberry resistance to anthracnose, shedding light on controlling strawberry anthracnose. In this study, we investigated the mechanisms underlying the enhanced strawberry resistance to anthracnose induced by P. fluorescens K1-3-1 by transcriptomic analysis. In addition, marker genes targeting for evaluation of P. fluorescens K1-3-1 inoculation were identified. The results showed that 44 genes were differentially expressed in the strawberry seedlings pretreated with P. fluorescens K1-3-1, when compared to the cotrol groups. Sixteen genes were selected as candidate marker genes, among which five were specifically induced by P. fluorescens K1-3-1, indicating their potential as marker genes. Several genes involved in defense responses were induced in the micropropagated strawberry seedlings pretreated with P. fluorescens K1-3-1 for two days. The data of RNA-seq analysis also revealed that after 7-day acclimatization, the genes involved in chromatin assembly were induced by P. fluorescens K1-3-1. Previous studies showed that PGPMs could prime plant immunity through chromatin modification, resulting in faster and stronger immune responses under stresses. Compared to the mock inoculation group, the expressions of the representative defense genes in response to C. siamense ML133, such as NPR1, WRKY70, PR1, and PR5, were induced more rapidly, stronger or longer in the strawberry seedlings pretreated with P. fluorescens K1-3-1 within 72 hour post inoculation of C. siamense ML133. Taking together, it is likely that P. fluorescens K1-3-1 could induce priming effects via chromatin remodeling, leading to enhanced strawberry resistance to anthracnose. Further investigation of related mechanisms could be carried out in the future.