Quantification of fungal growth by qPCR

Fungal spores production and infection:

Botrytis cinerea was cultivated on potato dextrose plates at 22℃ for 10 days. Spores were collected, washed, and frozen at -80 °C in 0.8% NaCl at a concentration of 10⁷ spores mL^-1. 

For droplet inoculation of Arabidopsis plants, 2 µL of 2.5*10⁵ spores mL^-1 was applied to single leaves of 4-week-old intact plants. For mock treatment, buffer alone was used. Plants were kept prior to and during the infection under sealed hoods under high humidity. 

Quantification of fungal biomass relative to plant biomass by qPCR was basically performed as previously described with some modification as follow (Gachon and Saindrenan, 2004). 

Fungal growth quantification:

1. Leaves of the indicated Arabidopsis lines were inoculated with two 2-μL droplets of B. cinerea spores. 3 days later, whole leaves with similar weight were harvested for DNA extracted. 

2. Plant samples were homogenized using a TissueLyser (Qiagen, Hilden, Germany) for 2×30 s at 30 strokes/s.

3. A total of 400 µL of DNA extraction bufer (200 mM Tris–HCl, pH 7.5; 250 mM NaCl; 25 mM EDTA, and 0.5% SDS) was added to each of the homogenized samples, which were shaken again in the Tissue Lyser for 10 s at 30 strokes/s. 

4. DNA extractions were then performed as commonly described and same amount of elution buffer was finally added to dissolve DNA. For each DNA sample, at least three technical replicates were performed.  

5. Quantitative real‑time PCR. 2uL of DNA was mixed with 0.4 mM gene-specifc primers (Cutinase A was used for B. cinerea, while SKII was used for Arabidopsis) and SYBR Green Supermix in a total volume of 25 µL. The reaction mixture contained 12.5 µL of SYBR Green, 1.5 µL of the forward and reverse primers, 9 µL of ddH2O and 2 µL of template DNA. qPCR was performed using the Applied Biosystems 7500 Sequence Detection System (ABI, Massachusetts, USA). The PCR program consisted of a preliminary step of 1 min at 95 °C followed by 40 cycles at 95 °C for 15 s and 60 °C for 34 s. The no template control for each primer pair was included in each run. 

6. The results were analysed using the Applied Biosystems 7500 software and Microsoft Office Excel based on the CT values observed. For each treatment, one representative set of result was presented as the mean 2^−∆∆CT value ± SEM. The amplifcation ratio of Cutinase A / SKII was then examed.

Reference:

Gachon C, Saindrenan P. (2004) Real-time PCR monitoring of fungal development in Arabidopsis thaliana infected by Alternaria brassicicola and Botrytis cinerea. Plant Physiol Biochem 42, 367-371.