Deciphering the Bacterial Microbiome of Citrus Plants in Response to ‘Candidatus Liberibacter asiaticus’-Infection and Antibiotic Treatments

Huanglongbing (HLB) is a worldwide devastating disease of citrus. There are no effective control measures for this newly emerging but century-old disease. A powerful oligonucleotide microarray of high-density 16S rRNA genes, the PhyloChip microarray, has been developed and effectively used to study bacterial diversity, especially from environmental samples. In this article, we aim to decipher the bacterial microbiome in HLB-affected citrus versus non-infected citrus as well as in citrus plants treated with ampicillin and gentamicin using PhyloChip-based metagenomics. The antibiotic treatments were conducted on the randomized complete block design with three replicates. For each replicate, 15 scions were treated in each antibiotic treatment (Amp and Gm) and control (CK1 and CK2). HLB-affected budsticks were sampled from severely HLB-affected field rough lemons (cv. Lemon #76) at the USDA-ARS-USHRL farm in Fort Pierce, FL and tested positive for Las by real-time qPCR. They were soaked in the antibiotic treatments; ampicillin sodium at a concentration of 1.0 g/L (Amp, Sigma-Aldrich, St. Louis, MO) or gentamicin sulfate at a concentration of 100 mg/L (Gm, Sigma-Aldrich, St. Louis, MO) and water as the diseased control (CK1), overnight in a fume hood under ventilation and lighting. Las-free budsticks, which tested negative by qPCR from healthy rough lemons, were also soaked in water as the healthy control (CK2). The budsticks were grafted onto two-year-old healthy grapefruit (Citrus paradisi 'Duncan') rootstocks and covered using plastic tape for three weeks. To improve scion growth, new flush from the rootstocks was removed after grafting and then allowed to grow. All experimental plants were grown in an insect-proof greenhouse. The first leaf samples from scions (rough lemon) and rootstocks (grapefruit) for DNA extraction were taken four months after inoculation, and second samplings were taken at six month after inoculation. The leaves were washed in tap water and then rinsed three times with sterile water. The midribs of the leaves were excised, frozen in liquid nitrogen, and stored at -80°C. The midribs of five leaves from each sample were pooled, and DNA was isolated for qPCR analysis for Las bacterium. DNA from the leaf midribs of scions for the PhyloChipT G3 analysis, which was extracted from all samples of the same treatment, was pooled in equal amounts and quantified by the PicoGreen® method. The PhyloChipTM G3 analysis was conducted by Second Genome Inc. (San Francisco, CA).

柑橘黄龙病（Huanglongbing, HLB）是一种全球性毁灭性柑橘病害。针对这一已出现百年之久的新兴病害，目前尚无有效防治措施。研究人员已开发出一款针对16S rRNA基因的高密度高效寡核苷酸微阵列（oligonucleotide microarray）——系统芯片微阵列（PhyloChip microarray），并将其有效应用于细菌多样性研究，尤其是环境样本中的细菌多样性分析。本研究旨在利用基于系统芯片微阵列的宏基因组学（metagenomics）技术，解析受黄龙病侵染的柑橘与健康柑橘，以及经氨苄青霉素（ampicillin）和庆大霉素（gentamicin）处理的柑橘植株中的细菌微生物组。本研究采用随机完全区组设计（randomized complete block design），设置3次生物学重复（replicates）。每一次重复中，氨苄青霉素（Amp）、庆大霉素（Gm）处理组及对照组（CK1、CK2）均接种15个接穗（scions）。受黄龙病侵染的芽条（budsticks）采自美国佛罗里达州皮尔斯堡USDA-ARS-USHRL农场中严重发病的粗柠檬（品种：Lemon #76），经实时定量PCR（real-time qPCR）检测呈Las阳性。将这些芽条在通风橱（fume hood）中经通风光照条件下过夜浸泡于不同处理液中：浓度为1.0 g/L的氨苄青霉素钠（Amp, Sigma-Aldrich, St. Louis, MO）、浓度为100 mg/L的硫酸庆大霉素（Gm, Sigma-Aldrich, St. Louis, MO），以及无菌水作为病健对照（CK1）；同时采集经实时定量PCR检测呈Las阴性的健康粗柠檬芽条，浸泡于无菌水中作为健康对照（CK2）。将上述芽条嫁接至2年生健康柚‘邓肯’（Citrus paradisi 'Duncan'）砧木上，并用塑料绑带（plastic tape）包裹固定3周。嫁接后需移除砧木萌发的新梢以促进接穗生长，待其恢复后正常培育。所有试验植株均种植于防虫温室（insect-proof greenhouse）中。分别于接种后4个月和6个月采集接穗（粗柠檬）和砧木（柚）的叶片样本用于DNA提取（DNA extraction）。采集的叶片先用自来水冲洗，再经无菌水冲洗3次。切除叶片的叶脉中脉（midribs），置于液氮（liquid nitrogen）中速冻后保存于-80℃冰箱。每份样本混合5片叶片的中脉，提取DNA用于Las细菌的实时定量PCR检测。将同一处理组所有样本的接穗叶片中脉DNA等量混合，采用皮克绿（PicoGreen®）法定量后，交由第二基因公司（Second Genome Inc., San Francisco, CA）进行系统芯片微阵列G3（PhyloChip™ G3）分析。