Transcriptome Analysis of Monilinia fructicola Following Exposure to Respiration Inhibitor Boscalid
Two isolates of Monilinia fructicola, MD132 and MD133 were used for this study. They were characterized recently for their fungicide resistance phenotype showing sensitive to boscalid and were stored on dried filter paper prior to this study. Starter colonies were initiated on Potato Dextrose Agar (PDA) at 22c. After three days, mycelium from advancing margins placed on peach fruit to obtain spores. Mature fruit were washed with soap to remove any pesticide residue and rinsed twice with sterile water. Fruit was then immersed in 10% bleach for 2 minutes to eliminate potential biological contaminants and again rinsed twice with sterile water. Peaches were then dried under a laminar flow hood. A PDA plug containing actively growing mycelium was placed on the fruit surface. For each isolate, three peaches were inoculated used as biological replicates (MD132a, MD132b, MD132c and MD133a, MD133b, MD133c). The fruit were kept individually in plastic boxes for 24 h and then incubated without boxes for another 8 days under the laminar flow hood with continuous florescent light. After 8 days, the conidia were gently scraped off with a sterile, disposable 10 µl inoculation loop (VWR international LLC, Radnor, PA) without touching the peach. The spores were suspended in potato dextrose broth (PDB) and adjusted to 1 to 2 × 10^5 spores/ml. Aliquots of 1.5 ml of spore suspension from each peach were shaken in 2 ml Eppendorf tubes at room temperature at 150 rpm for 15 h before fungicide treatment.    Commercial formulations of boscalid (Endura fungicide 70% W/W BASF) were used in this study at a final concentration of 20 μl/ml. The fungicide-treated spore suspensions were incubated at room temperature at 150 rpm for an additional 30 minutes(MD132ad, MD132bd, MD132cd and MD133ad, MD133bd, MD133cd). The tubes were then spun at 12,000 rpm for 5 minutes, the supernatant was discarded, and the germling pellet was rinsed with sterile water. This step was repeated twice before the pellets were ground in liquid nitrogen for RNA extraction.     RNA was isolated with the Qiagen RNeasy Plant Mini kit (cat# 74903, Qiagen, USA) from approximately 30 mg of ground tissue. Purity and concentration of the samples were estimated with a NanoDrop ND-8000 spectrophotometer (NanoDrop technologies Inc, USA), and the integrity of the RNA was evaluated on an Agilent RNA 6000 Nano LabChip using Agilent 2100 Bioanalyzer (Agilent Technologies, Germany). Purification of RNA was conducted using Illumina TruSeq RNA Sample Preparation Kit (Illumina, Inc., CA). To construct the 12 cDNA libraries, double-stranded cDNA was prepared with 1.5 µg of total RNA as mentioned above and sequenced on an Illumina HiSeq 2000 (PE) with 2 x 100 bp end read length (David H. Murdock Research Institute, NC).