tissue: midrib of leaf genotype: citrus unique hybrid, 10c-5-58 age: HLB-affected, 7-year-old citrus trees
Treatment protocol
The antibiotic treatments were conducted on the randomized complete block design with four replicates. For each replicate, five HLB-affected, 7-year-old citrus trees (a unique hybrid, 10c-5-58, which is an open-pollinated seedling from the combination of Lee mandarin × Orlando tangelo) at the USHRL farm, 10 cm in diameter, were injected with either 100 ml of the antibiotic combination treatment PS (5 g of penicillin G potassium + 0.5 g of Streptomycin per tree) or the antibiotic treatment KO (2 g of oxytetracycline + 1.0 g of kasugamycin per tree). Five trees were injected with water as injection controls (CK). Injections were made using an Avo-Ject syringe injector (a catheter-tipped 60 ml syringe; Aongatete Coolstores Ltd., NZ) beginning in August of 2010. The tapered tip was firmly fitted into a 19/64-in (7.5 mm) diameter hole, ≈3 cm deep, drilled into the tree. The injector was kept in the tree and the treatment lasted for one week in each injection-trunk. Treatments were repeated every 2 months for one year and ceased in August of 2011.
Growth protocol
HLB-affected, 7-year-old citrus trees (a unique hybrid, 10c-5-58, which is an open-pollinated seedling from the combination of Lee mandarin × Orlando tangelo) were planted at the USHRL farm, Fort Pierce, FL.
Extracted molecule
genomic DNA
Extraction protocol
Qiagen Plant Mini extraction of total DNA was performed according to the manufacturer's instructions.
Label
biotin
Label protocol
The 16S rRNA amplicons and a mixture of amplicons at known concentrations were combined, fragmented using DNAseI (Invitrogen, Carlsbad, CA), and biotin-labeled using the recommended protocol for Affymetrix Prokaryotic Arrays.
Hybridization protocol
Labeled products were hybridized with PhyloChip™ Array, version G3, overnight at 48°C and 60 rpm.
Scan protocol
PhyloChip arrays were washed, stained, and scanned using a GeneArray® scanner (Affymetrix). Each scan is captured using standard Affymetrix software (GeneChip® Microarray Analysis Suite).
Description
Hyberscore data from PS-treatment at time point 3
Data processing
Samples are processed in a Good Laboratory Practices (GLP) compliant service laboratory running Quality Management Systems for sample and data tracking. The laboratory implements detailed SOPs, equipment and process validation, training, audits and document control measures. QC and QA metrics are maintained for all sample handling, processing and storage procedures.Details on probe selection, probe scoring, data acquisition, and preliminary data analysis are presented in Hazen et al. (2010) and premilary analyses were performed by Second Genome (San Bruno, CA, USA). In brief, two criteria were met when the probe pairs scored as positive: (i) the PM (Perfect Match) probe’s intensity of fluorescence was greater than 1.3 times that from the MM (Mismatch) control and (ii) the difference in intensity, PM minus MM, was at least 500 times greater than the squared noise value (>500 N2), which was the variation in pixel intensity signals observed by the scanner as it read the array surface. An OTU was considered present in the sample when over 90% of its assigned probe pairs were positive. A hybridization intensity score (HybScore) was calculated in arbitrary units for each probe set as the trimmed average (maximum and minimum values removed before averaging) of the PM minus MM intensity differences across the probe pairs in a given probe set. The values of the present OTUs used for each taxa-sample intersection were populated in two distinct ways. In the first case, the abundance metrics were used directly (AT). In the second case, binary metrics were created where 1’s represented presence, 0’s indicated absence (BT).
Characterization of the microbial community structure in Candidatus Liberibacter asiaticus-infected Citrus plants treated with antibiotics in the field
