Sequencing and enrichment of Candidatus Liberibacter asiaticus from its psyllid host using filtration and host DNA removal

A barrier to cost-effective sequence analysis of obligately biotrophic bacterial mutualists or pathogens is host DNA contamination. The pathogen 'Candidatus Liberibacter asiaticus' (CLas), whose growth in a variety of citrus species results in citrus greening disease (huanglongbing), is a prime example of a bacterium whose host-associated lifestyle has presented a significant challenge to generating genome sequences for comparative genomic analyses or antibiotic resistance screening. In addition to citrus, CLas also replicates within and is transmitted by the Asian citrus psyllid, Diaphorina citri, to citrus when the insect feeds. While methods to enrich and sequence CLas from the Asian citrus psyllid exist, they are laborious, costly, and result in biases (e.g., during multiple displacement amplification) that limit the utility of the resulting genomes for downstream use. To overcome these issues, we've developed a simple homogenization, filtration, and DNase-treatment methodology to directly sequence CLas from individual Diaphorina citri adults and have shown that CLas comprises upwards of 60% of the total sequencing reads from these samples. Furthermore, enrichment of other D. citri bacterial endosymbionts was achieved, highlighting the applicability of this approach as a simple yet comprehensive method to simultaneously explore multiple host-associated bacterial populations. While CLas enrichment and sequencing was more effective in lab-reared D. citri individuals (in which CLas reaches high titers), we leveraged this method to sequence DNA from wild-caught D. citri individuals from a Florida citrus grove and were able to sequence and assemble nearly complete citrus mitochondrial and plastid genomes, fungal mitochondrial genomes, and bacterial genomes associated with wild D. citri populations. Hence, results from the application of this method can directly inform agricultural management strategies involving surveillance of insect populations related to food preferences and their microbiomes.专性活营养细菌共生体或致病菌（obligately biotrophic bacterial mutualists or pathogens）的低成本高效测序分析面临的一大核心障碍为宿主DNA污染。致病菌‘Candidatus Liberibacter asiaticus’（CLas）便是典型案例：其在多种柑橘属植物中增殖会引发柑橘黄龙病（huanglongbing），而这种宿主关联的生活方式极大阻碍了用于比较基因组分析或抗生素抗性筛选的全基因组测序工作。除柑橘宿主外，CLas还会在亚洲柑橘木虱（Diaphorina citri）体内增殖，并在该昆虫取食时将病菌传播至柑橘植株。尽管目前已存在从亚洲柑橘木虱中富集并测序CLas的方法，但这些方法操作繁琐、成本高昂，且会引入偏差（例如在多重置换扩增（multiple displacement amplification）过程中），导致所得基因组在后续研究中的应用受限。为解决上述问题，本研究开发了一套简易的均质化、过滤及脱氧核糖核酸酶（DNase）处理流程，可直接从单个成年亚洲柑橘木虱样本中测序CLas，实验结果显示CLas在这些样本的总测序读段中占比可达60%以上。此外，该方法还可实现对亚洲柑橘木虱体内其他细菌内共生体的富集，证明此方法作为一种简易且全面的手段，可同时探究多种宿主关联的细菌种群。尽管CLas的富集与测序在实验室饲养的亚洲柑橘木虱个体（此类样本中CLas滴度较高）中效果更佳，但本研究仍利用该方法对佛罗里达州柑橘园捕获的野生亚洲柑橘木虱个体的DNA进行了测序，成功组装得到近乎完整的柑橘线粒体基因组、质体基因组、真菌线粒体基因组以及与野生亚洲柑橘木虱种群相关的细菌基因组。因此，该方法的应用结果可直接为农业管理策略提供参考，包括针对昆虫种群取食偏好及其微生物组的监测工作。