With the current advancements in DNA sequencing technology, the limiting factor in long-read metagenomic assemblies is now the quantity and quality of input DNA. Although these requirements can be met through the use of axenic bacterial cultures or large amounts of biological material, insect systems that contain unculturable bacteria or that contain a low amount of available DNA cannot fully utilize the benefits of third-generation sequencing. The citrus greening disease insect vector Diaphorina citri is an example that exhibits both of these limitations. Although endosymbiont genomes have mostly been closed after the short-read sequencing of amplified template DNA, creating de novo long-read genomes from the unamplified DNA of an insect population may benefit communities using bioinformatics to study insect pathosystems. Here all four genomes of the infected D. citri microbiome were sequenced to closure using unamplified template DNA and two long-read sequencing technologies. Avoiding amplification bias and using long reads to assemble the bacterial genomes allowed for the circularization of the Wolbachia endosymbiont of Diaphorina citri for the first time and paralleled the annotation context of all four reference genomes without utilizing a traditional hybrid assembly. The strategies detailed here are suitable for the sequencing of other insect systems for which the input DNA, time, and cost are an issue.

Citrus greening disease is now completely endemic to Florida citrus trees, having spread rapidly across all counties in the past 20 years and causing devastating economic losses. The disease's etiological agent is the phloem-restricted bacterium ‘ Candidatus Liberibacter asiaticus’ ( CLas). Liberibacter crescens, the closest culturable relative, was discovered to prefer citrate as its most effective carbon and energy source. Citrus plants load free citrate in response to phosphorus deficiency. In Florida's calcareous soils, supplemental phosphate fertilization is very low due to the assumption that phosphate is readily available for plants through the soil. It is likely that citrus trees are loading citrate to mine phosphorus from the soil, which could inadvertently exacerbate CLas infection. In greenhouse experiments, foliar-sprayed phosphate is taken up by the plants, resulting in decreased free-citrate levels and changes in fumaric and succinic acids in the phloem, in addition to delaying titer detection of CLas. Our field experiments—where mature trees have a well-established huanglongbing infection maintained by infected psyllids—exhibited a lower CLas titer for some of the phosphate treatments across time.