Endophytic colonization of P. chlamydosporia was verified by qPCR. The roots were collected and stored at −80 °C. Total DNA was extracted from around 100 mg of tissue using a plant/fungi DNA isolation kit (Norgen Biotek, Thorold, ON, Canada) following the manufacturer’s instructions. Total DNA was quantified by a Nanodrop™ spectrophotometer (Thermo Fisher Sc., MA, USA), and a 50 ng/µL concentration solution was prepared for PCR reactions. For P. chlamydosporia detection and quantification, qPCR experiments were conducted using an Aria PCR device (Agilent Scientific Instruments, Santa Clara, CA, USA). The alkaline serine protease (VCP1) was selected as the target gene using primers For0 and Rev0 [15 (link)]. Amplification reactions were performed in a 15 µL volume with 2× SYBR green master mix (AllGene, Madison, WI, USA), 50 ng of total DNA and 500 nM of primer. The thermal profile was 95 °C for 3 min, 40 cycles at 95 °C for 30 s, 52 °C for 20 s, and 72 °C for 10 s. A standard curve was constructed using serial dilutions from 1 ng to 1 pg of genomic DNA of the P. chlamydosporia DSM 26985. The initial amount of fungal DNA contained in total root DNA was calculated by the correlation of quantification cycle (Cq) values with Cq values in the standard curve. Data significance was evaluated by applying Student’s t-test (p ≤ 0.05).
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