外源性抗菌胜肽LsGRP1C抗草莓炭疽病之機制研究

Abstract

Colletotrichum spp.引起炭疽病使草莓產量受嚴重衝擊，過度依賴化學藥劑防治所衍生出的抗藥性及農藥殘留問題，又是草莓產業一大隱憂，開發新穎的天然源防治資材即為當前重要課題。抗菌胜肽被認為是可做為對抗微生物感染的新興防治資材。LsGRP1 (Lilium ‘Star Gazer’ glycine rich protein 1)是葵百合上的防禦蛋白，已知LsGRP1 C端區域 (LsGRP1C)，是一富含半胱胺酸的抗菌區段，能有效抑制多種植物病原真菌孢子發芽且對多種細菌有致死作用。本研究以化學合成LsGRP1C對草莓炭疽病菌進行生體外的抑菌機制探討，以及於草莓植株上進行抑病機制研究。生體外抑菌試驗結果顯示，以無抑菌效力濃度的金屬螯合劑乙二胺四乙酸(ethylenediaminetetraacetic acid disodium salt dihydrate, EDTA)共同使用，可增強LsGRP1C對不同種Colletotrichum spp.的抑制作用。組織化學染色則證實在加入EDTA後，LsGRP1C能誘使Colletotrichum sp. SWHL-1產生類細胞凋亡與細胞膜滲漏的現象。值得注意的是，植體上原位保護試驗顯示，於草莓的葉、莖或果實單獨處理LsGRP1C，即可有效減少甚至完全抑制壞疽病斑的產生。進一步以Colletotrichum siamense ML133進行草莓葉片上LsGRP1C的抑病機制研究，顯示LsGRP1C在不直接與病原菌接觸下，即可使C. siamense ML133引起之病徵嚴重程度降低；LsGRP1C的處理葉及系統葉經flg22刺激均可增加癒傷葡聚醣累積。因此，推測LsGRP1C除了直接抑制病原菌生長，尚能透過引發植物誘導抗病反應的作用模式，以抑制草莓炭疽病的發生。

Anthracnose caused by Colletotrichum spp. seriously affects strawberry production. However, heavy dependence of pesticide in strawberry industry often leads to the resistant pathogens and chemical residue problem, so the development of novel control agents from natural resources becomes an important issue. Antimicrobial peptides (AMPs) are the emerging disease management materials to combat microbial infection. LsGRP1 (Lilium ‘Star Gazer’ glycine rich protein 1) is a defense protein of Lilium ‘Star Gazer’. The cysteine-rich C-terminal region, LsGRP1C, exhibits antimicrobial activity to inhibit spore germination of various plant pathogenic fungi and confer lethal effect on a variety of bacteria. In this study, the synthetic LsGRP1C was used in vitro to dissect its antifungal activities on Colletotrichum spp., and in planta to investigate the disease suppression function on strawberry anthracnose. In vitro antifungal assays revealed that the co-treatment of ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA), a chelating agent, at a concentration without antifungal activity, enhanced the inhibitory effect of LsGRP1C on different species of Colletotrichum spp. Histochemical staining demonstrated that the mixture of LsGRP1C and EDTA caused apoptosis-like programmed cell death and membrane permeabilization of Colletotrichum sp. SWHL-1. Worth to notice, in situ protection assays showed that treatment with LsGRP1C alone significantly inhibited necrotic symptom development in leaves, stem and fruits of strawberry. Furthermore, LsGRP1C could systemically enhance the leaf resistance to Colletotrichum siamense ML133. LsGRP1C treated leaves and system leaves could increase the accumulation of callose by flg22 stimulation. Callose deposition also could be triggered by flg22 in the systemic leaves. Herein, LsGRP1C is strongly speculated to combat strawberry anthracnose not only via inhibiting pathogen growth but also has a action mode of inducing plant defense.