Table_1_Bacterial Seed Endophytes of Domesticated Cucurbits Antagonize Fungal and Oomycete Pathogens Including Powdery Mildew.XLSX

The cucurbit vegetables, including cucumbers, melons and pumpkins, have been cultivated for thousands of years without fungicides. However, their seed germination stage is prone to be infected by soil-borne fungal and oomycete pathogens. Endophytes are symbionts that reside inside plant tissues including seeds. Seed endophytes are founders of the juvenile plant microbiome and can promote host defense at seed germination and later stages. We previously isolated 169 bacterial endophytes associated with seeds of diverse cultivated cucurbits. We hypothesized that these endophytes can antagonize major fungal and oomycete pathogens. Here we tested the endophytes for in vitro antagonism (dual culture assays) against important soil-borne pathogens (Rhizoctonia solani, Fusarium graminearum, Phytophthora capsici, Pythium aphanidermatum). The endophytes were also assayed in planta (leaf disk and detached leaf bioassays) for antagonism against a foliar pathogen of global importance, Podosphaera fuliginea, the causative agent of cucurbit powdery mildew. The endophytes were further tested in vitro for secretion of volatile organic compounds (VOCs) known to induce plant defense. Extracellular ribonuclease activity was also tested, as a subset of pathogenesis-related (PR) proteins of plant hosts implicated in suppression of fungal pathogens, displays ribonuclease activity. An unexpected majority of the endophytes (70%, 118/169) exhibited antagonism to the five phytopathogens, of which 68% (50/73) of in vitro antagonists belong to the genera Bacillus and Paenibacillus. All Lactococcus and Pantoea endophytes exhibited anti-oomycete activity. However, amongst the most effective inoculants against Podosphaera fuliginea were Pediococcus and Pantoea endophytes. Interestingly, 67% (113/169) of endophytes emitted host defense inducing VOCs (acetoin/diacetyl) and 62% (104/169) secreted extracellular ribonucleases in vitro, respectively. These results show that seeds of cultivated cucurbits package microbes with significant disease-suppression potential. As seeds can act as vectors for genetic transmission of endophytes across host generations, it is interesting to hypothesize whether humans, when selecting seeds of healthy hosts, may have inadvertently selected for disease-suppressing seed endophytes. As the majority of pathogen-suppressing endophytes belong to Bacillus and Paenibacillus, and since Bacilli are widely used as commercial biocontrol agents of vegetables, we propose that these agents are mimicking the ecological niche established by their endophytic cousins.

葫芦科蔬菜（涵盖黄瓜、甜瓜与南瓜）已有数千年无杀菌剂施用的栽培历史。然而，其种子萌发阶段极易遭受土传真菌及卵菌病原菌的侵染。内生菌（endophytes）是定殖于植物组织（包含种子）内的共生体；种子内生菌是植物幼苗微生物组的奠基者，可在种子萌发及后续生长阶段促进宿主防御。此前我们从多种栽培葫芦科植物的种子中分离得到169株细菌内生菌，并提出假说：这些内生菌能够拮抗主要的真菌与卵菌病原菌。本研究通过体外拮抗试验（双培养测定法），检测了这些内生菌对4种重要土传病原菌——立枯丝核菌（Rhizoctonia solani）、禾谷镰孢菌（Fusarium graminearum）、辣椒疫霉（Phytophthora capsici）及瓜果腐霉（Pythium aphanidermatum）的拮抗活性。同时，通过植物体内试验（叶盘与离体叶片生物测定），评估了内生菌对全球性分布的重要叶部病原菌——瓜类单囊壳菌（Podosphaera fuliginea，葫芦科白粉病的致病菌）的拮抗效果。此外，我们还在体外检测了内生菌分泌可诱导植物防御的挥发性有机化合物（VOCs）的能力，并检测了其细胞外核糖核酸酶活性——植物宿主的部分病程相关（PR）蛋白具备抑制真菌病原菌的功能，且本身具有核糖核酸酶活性。出人意料的是，70%（118/169）的内生菌对5种植物病原菌表现出拮抗活性，其中68%（50/73）的体外拮抗菌株归属于芽孢杆菌属（Bacillus）和类芽孢杆菌属（Paenibacillus）。所有乳球菌属（Lactococcus）和泛菌属（Pantoea）内生菌均表现出抗卵菌活性；而对瓜类单囊壳菌防控效果最佳的接种剂则为片球菌属（Pediococcus）和泛菌属内生菌。值得注意的是，分别有67%（113/169）的内生菌可分泌诱导宿主防御的VOCs（乙酰甲基甲醇/双乙酰），62%（104/169）的内生菌可在体外分泌细胞外核糖核酸酶。上述研究结果表明，栽培葫芦科植物的种子携带有具备显著病害抑制潜力的微生物菌群。由于种子可作为内生菌在宿主世代间进行遗传传递的载体，我们不禁提出一个有趣的假说：人类在筛选健康植株的种子时，是否无意间选择了具备病害抑制能力的种子内生菌？鉴于多数具有病原菌拮抗活性的内生菌归属于芽孢杆菌属和类芽孢杆菌属，且芽孢杆菌已被广泛用作商业化蔬菜生防制剂，我们推测这些商业化生防制剂模拟了其内生同源菌种所占据的生态位。