Plant fungi dna isolation kit

Botrytis cinerea spores were diluted to 5 × 10⁴ spores per milliliter in Vogel buffer (Vogel, 1956 ) for drop inoculation assay or 2 × 10⁵ spores per milliliter in Vogel buffer for spray inoculation assay used for RT-qPCR analysis. The spores were pregerminated for 4 h before inoculations were carried out. For drop inoculations, 6 µL of spore suspension in Vogel buffer was carefully placed on the adaxial side (away from the midrib) of a fully expanded rosette leaf of 6–7 week-old Arabidopsis plants (at least 30 leaves were used from 10 independent plants). For spray inoculation, plants were sprayed until droplets began to run off the leaves (Mengiste et al., 2003 (link)). Inoculated plants were covered and grown under high humidity conditions for 3 or 4 days. Lesion diameters of drop-inoculated leaves were measured using a digital caliper and used to calculate lesion diameter. Spray-inoculated rosette leaves were harvested at 3 dpi. For fungal DNA quantification, fungal DNA was extracted using a plant/fungi DNA isolation kit (Norgen Biotek Corp, Thorold, Ontario, Canada) following the manufacturer’s protocol. The fungal β-ACTIN genomic DNA was quantified by qPCR (Ettenauer et al., 2014 (link)) using primers listed in Supplemental Table S1.

Genomic DNA was extracted from ‘VABM29’, ‘Bounty’, ‘Glowing Star’ and ‘V85331’ leaves using a Plant/Fungi DNA Isolation Kit (Norgen, Thorold, ON, Canada). The 5’ upstream region of PpJAZ1 was isolated using the Universal Genome Walker Kit (Clontech, Palo Alto, CA, USA). Nested PCR reactions were performed using reverse primers in the coding region (see Additional file 6: Table S5) and adaptor primers provided by the manufacturer. PCR-amplified fragments were cloned and sequenced as explained above. Promoter sequences were analyzed using the Plant Promoter Analysis Navigator (PlantPAN) [68 ].

The extraction of DNA was performed from mycelium using the Plant/Fungi DNA isolation kit (Norgen Biotek Corp., Canada), following the manufacturer’s instructions, or manually, using the cetyl-trimethyl ammonium bromide procedure (CTAB). The cells were obtained from pure in vitro cultures maintained in Petri dishes with PDA medium, and for the CTAB extraction only, they were fragmented in a vortex with a few, 400 µm diam. glass beads added (Sigma-Aldrich, St. Louis, MO, USA), for 5 min.

Plant roots were collected at the last time-point assay and conserved at −80 °C. Total DNA was extracted from around 100 mg of tissue using a plant/fungi DNA isolation kit (Norgen Biotek) following the manufacturer’s instructions. Total DNA was quantified by nanodrop and a 50 ng/µL concentration solution was prepared for amplification reactions. For P. chlamydosporia detection and quantification, qPCR experiments were conducted in an Aria (Agilent Scientific Instruments, Santa Clara, CA, USA) PCR device with VCP1 as the target gene, using the primers For0, (5′-ctcgaggctgcccaac) and Rev0 (5′-tgcatgcactaggctcgg) [41 ]. Amplification reactions were performed in a 15 µL volume with 2 x SYBR green master mix (AllGene), 50 ng of total DNA and 500 nM of primer. The thermal profile was 95 °C for 3min, 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 P. chlamydosporia. The initial amount of fungal DNA contained in total root DNA was calculated by correlation of quantification cycle (Cq) values with Cq values in the standard curve. Data significance were evaluated by applying Student’s t-test (p ≤ 0.05).

DNA of the pathogen isolated from infected root tissues was extracted from mycelium cultured for 14 days on PDA plates at 25 °C. DNA extractions were performed using the plant/fungi DNA isolation kit (Norgen Biotek Corp., Thorold, Canada). PCR reactions contained reaction buffer, 2.2 mM MgCl₂, 200 µM of each dNTP, 2.5 unit of Taq DNA polymerase, a 30-ng DNA template and 50 pmol of each primer, making to a final volume of 50 µL. PCR was set for 32 cycles; each cycle consisted of: 94 °C for 1 min; 58 °C for 1 min; 72 °C for 1 min. For the three Fusarium spp., PCR amplified target regions of ITS using ITS1 and ITS4 primers [56 ], 28S rDNA using LRoR and LR5 [57 (link)], TEF1-α using EF1-728F and EF1-986R [58 (link)], and β-tubulin using Bt1a and Bt1b [59 (link)]. In addition, pFalb11 and pFalb28 belonging to F. oxysporum f. sp. albedinis were also amplified. All primer sequence sets can be found in Table S1.

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).