Department of Plant Pathology & Environmental Microbiology, The Pennsylvania State University, University Park, Pennsylvania, USA.
Metabolomics Core Facility, Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, Pennsylvania, USA.
Appl Environ Microbiol. 2019 Jul 18;85(15). doi: 10.1128/AEM.00935-19. Print 2019 Aug 1.
The Gram-negative bacterium causes fire blight disease of apples and pears. While the virulence systems of have been studied extensively, relatively little is known about its parasitic behavior. The aim of this study was to identify primary metabolites that must be synthesized by this pathogen for full virulence. A series of auxotrophic mutants, representing 21 metabolic pathways, were isolated and characterized for metabolic defects and virulence in apple immature fruits and shoots. On detached apple fruitlets, mutants defective in arginine, guanine, hexosamine, isoleucine/valine, leucine, lysine, proline, purine, pyrimidine, sorbitol, threonine, tryptophan, and glucose metabolism had reduced virulence compared to the wild type, while mutants defective in asparagine, cysteine, glutamic acid, histidine, and serine biosynthesis were as virulent as the wild type. Auxotrophic mutant growth in apple fruitlet medium had a modest positive correlation with virulence in apple fruitlet tissues. Apple tree shoot inoculations with a representative subset of auxotrophs confirmed the apple fruitlet results. Compared to the wild type, auxotrophs defective in virulence caused an attenuated hypersensitive immune response in tobacco, with the exception of an arginine auxotroph. Metabolomic footprint analyses revealed that auxotrophic mutants which grew poorly in fruitlet medium nevertheless depleted environmental resources. Pretreatment of apple flowers with an arginine auxotroph inhibited the growth of the wild-type , while heat-killed auxotroph cells did not exhibit this effect, suggesting nutritional competition with the virulent strain on flowers. The results of our study suggest that certain nonpathogenic auxotrophs could have utility as fire blight biocontrol agents. This study has revealed the availability of a range of host metabolites to cells growing in apple tissues and has examined whether these metabolites are available in sufficient quantities to render bacterial synthesis of these metabolites partially or even completely dispensable for disease development. The metabolomics analysis revealed that auxotrophic mutants have substantial impact on their environment in culture, including those that fail to grow appreciably. The reduced growth of virulent on flowers treated with an arginine auxotroph is consistent with the mutant competing for limiting resources in the flower environment. This information could be useful for novel fire blight management tool development, including the application of nonpathogenic auxotrophs to host flowers as an environmentally friendly biocontrol method. Fire blight management options are currently limited mainly to antibiotic sprays onto open blossoms and pruning of infected branches, so novel management options would be attractive to growers.
革兰氏阴性细菌 引起苹果和梨的火疫病。虽然 的毒力系统已经被广泛研究，但对其寄生行为的了解相对较少。本研究的目的是鉴定该病原体完全毒力所必需合成的初级代谢物。一系列代表 21 种代谢途径的 营养缺陷型突变体被分离并表征，以研究其在苹果幼果和嫩枝中的代谢缺陷和毒力。在离体的苹果幼果上，与野生型相比，精氨酸、鸟嘌呤、己糖胺、异亮氨酸/缬氨酸、亮氨酸、赖氨酸、脯氨酸、嘌呤、嘧啶、山梨糖醇、苏氨酸、色氨酸和葡萄糖代谢缺陷型突变体的毒力降低，而天冬酰胺、半胱氨酸、谷氨酸、组氨酸和丝氨酸生物合成缺陷型突变体的毒力与野生型相同。在苹果幼果培养基中生长的营养缺陷型突变体的生长与在苹果幼果组织中的毒力呈适度正相关。用一组代表性的营养缺陷型突变体接种苹果树嫩枝，证实了苹果幼果的结果。与野生型相比，除精氨酸营养缺陷型外，在烟草中引起弱过敏免疫反应的毒力缺陷型突变体。代谢组学足迹分析显示，尽管在幼果培养基中生长不良的营养缺陷型突变体仍然耗尽了环境资源。用精氨酸营养缺陷型突变体预处理苹果花可抑制野生型 的生长，而热灭活的营养缺陷型细胞则没有表现出这种效应，这表明在花上与致病菌株存在营养竞争。我们的研究结果表明，某些非致病性 营养缺陷型突变体可能作为火疫病生物防治剂具有实用性。本研究揭示了在苹果组织中生长的 细胞可利用一系列宿主代谢物，并研究了这些代谢物是否存在足够的数量，以使细菌 对这些代谢物的合成部分或甚至完全不需要用于疾病的发展。代谢组学分析表明，营养缺陷型 突变体对其在培养中的环境有很大的影响，包括那些生长不明显的突变体。用精氨酸营养缺陷型处理过的花朵上的致病 的生长减少与突变体在花环境中竞争有限资源一致。这些信息可能对新型火疫病管理工具的开发有用，包括将非致病性 营养缺陷型应用于宿主花朵作为一种环保的生物防治方法。目前，火疫病的管理方法主要限于在开放的花朵上喷洒抗生素和修剪感染的树枝，因此新的管理方法对种植者很有吸引力。
