0 g of kasugamycin per tree) Five trees were injected with water

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 two months for one year and ceased in August of 2011. Before and during treatment more than 30 leaf samples per tree were taken from DNA Damage inhibitor different positions around the tree canopies for qPCR assays at two month intervals. Genomic DNA extraction and qPCR analysis for the HLB bacterium Each leaf sample was rinsed three times with sterile water. Midribs were separated from the leaf samples and cut into pieces of 1.0 to 2.0 mm. DNA was extracted from 0.1 g of tissue (fresh weight) of leaf midribs using Qiagen’s DNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. The bacterial titers were quantified by qPCR using the primers and probes Ilomastat clinical trial (HLBas, HLBr, and HLBp)

for ‘Ca. L. asiaticus’ as described previously [17, 33]. Data were analyzed by a generalized linear mixed model using the SAS procedure GLIMMIX. Differences among treatments and sampling time points were determined with the LINES option of the buy Belnacasan LSMEANS statement. PCR amplification of 16S rRNA genes for PhyloChip™ G3 hybridization DNA for the PhyloChip™ G3 analysis, which was extracted from 20 samples of the same treatment, was pooled in equal amounts and quantified by the PicoGreen® method. The PhyloChip™ G3 analysis was conducted by Second Genome Inc. (San

Francisco, CA). The bacterial 16S rRNA genes were amplified from the above pooled DNA using an learn more eight-temperature gradient PCR (annealing temperatures of 48.0, 48.8, 50.1, 51.9, 54.4, 56.3, 57.5, and 58.0°C) with bacterially directed primers 27 F (5-AGA GTT TGA TCC TGGCTC AG) and 1492R (5-GGT TAC CTT GTT ACG ACT T). In brief, the 25 μl reactions (final concentrations were 1× Ex Taq Buffer with 2 mM MgCl2, 200 nM each primer (27 F and 1492R), 200 μM each dNTP, 25 μg bovine serum albumin (Roche Applied Science, Indianapolis, IN), and 0.625 U Ex Taq (TaKaRa Bio Inc., Shiga, Japan) were amplified using an iCycler (Bio-Rad, Hercules, CA) under the following thermocycling conditions: 95°C for 3 min for initial denaturation, 35 cycles of 95°C for 30 s, 48 to 58°C for 30 s, and 72°C for 2 min, and then final extension for 10 min at 72°C. PCR products from each annealing temperature for a sample were combined and concentrated using Amicon centrifugal filter units (Millipore Corp., Billerica, MA). The samples were quantified by electrophoresis using an Agilent 2100 Bioanalyzer® before application to the PhyloChip™ G3 array. PhyloChip Control Mix™ was added to each amplified product.

Comments are closed.