Winter rye cover crops shelter competent squash phyllosphere bacteria to reduce Pseudomonas syringae populations and symptoms

Cover crops, a soil conservation practice, are amongst tools that can contribute to reducing disease pressure caused by Pseudomonas syringae, considered as one of the most important bacterial plant pathogens. Indeed, it has recently been demonstrated that phyllosphere (leaf surface) bacterial community structure changed when squash (Cucurbita pepo) is grown with a rye cover crop treatment, followed by a decrease of P. syringae symptoms. Application of biocontrol agents is a known agricultural practice in integrated pest management procedures to mitigate crop losses due to microbial disease. In this study, we pursue the hypothesis that some phyllosphere bacteria promoted when squash are grown on cover crops could be isolated and used as a biocontrol agent to decrease P. syringae symptoms. We grew squash during a two-year field experiment using four agricultural practices: bare soil, cover crops, chemically terminated cover crops and plastic cover. We sampled squash leaves at 3 different dates each year and constructed a bacterial collection of cultivable strains isolated from squash leaves and rye cover crop material. Each isolated strain was identified by 16S rRNA gene sequencing and subjected to an in vitro (Petri dish) and in vivo (greenhouse) competition assay against P. syringae. Four bacterial isolates belonging to the genera Acinetobacter, Pseudomonas, Delftia and Rhizobium were proven to inhibit P. syringae growth and symptom development. Strikingly, the antagonist impact on P. syringae was stronger on older leaves. This study shed light on the importance of bacterial isolation from different sources to promote disease control

覆盖作物（cover crops）作为一种土壤保育措施，是可减轻丁香假单胞菌（Pseudomonas syringae）引发的病害压力的有效手段之一，而丁香假单胞菌被视为最重要的植物细菌性病原物之一。近期已有研究证实，当西葫芦（Cucurbita pepo）采用黑麦覆盖作物处理种植时，其叶围（phyllosphere，即叶片表面）细菌群落结构会发生改变，伴随丁香假单胞菌病害症状的减轻。生物防治剂（biocontrol agents）的应用是有害生物综合治理（integrated pest management）流程中公认的农业措施，可减轻由微生物病害引发的作物产量损失。本研究提出如下假说：在覆盖作物种植模式下西葫芦叶围中增殖的部分细菌可被分离，并作为生物防治剂以减轻丁香假单胞菌的病害症状。本研究开展了为期两年的田间试验，设置四种种植模式：裸地、覆盖作物、化学终止的覆盖作物以及地膜覆盖。每年于三个不同时间节点采集西葫芦叶片样本，并构建了从西葫芦叶片和黑麦覆盖作物材料中分离得到的可培养细菌菌株库。所有分离得到的菌株均通过16S rRNA基因测序完成物种鉴定，并开展了针对丁香假单胞菌的体外（in vitro，培养皿）与体内（in vivo，温室）竞争试验。最终筛选得到4株分别属于不动杆菌属（Acinetobacter）、假单胞菌属（Pseudomonas）、代尔夫特菌属（Delftia）和根瘤菌属（Rhizobium）的细菌菌株，经证实可抑制丁香假单胞菌的生长与病害症状发展。值得注意的是，这些菌株对丁香假单胞菌的拮抗作用在老叶中更为显著。本研究揭示了从不同来源分离细菌以助力病害防控的重要价值。