Materials and Reagents

Freshly harvested tomatoes and apples (fully ripe)
Botrytis cinerea (B05.10): Supplied by Prof. Paul Tudzynski Westfaelische Wilhelms-Universitaet Muenster, Germany
KH₂PO₄ (Beijing Chemical Works, catalog number: HG/T 1274-1993 )
Glucose (Beijing Chemical Works, catalog number: HG/T 3475-1999 )
2% sodium hypochlorite (Xilong Chemical, catalog number: 7681-52-9 )
Potato
Dextrose
PDA medium (see Recipes)
2% sodium hypochlorite (see Recipes)
KH₂PO₄-glucose solution (see Recipes)

Equipment

Glass stick
Hemacytometer (QIUJING, model: XB-K-25 )
Cheesecloth
Vortexer (Select BioProducts, model: SBS100-2 )
Optical microscope (Chongqing Optec Instrument, model: B203LED )
Clean bench (Donglian Electronic & Technology Development, model: SCB-1520 )
Pipette
Plastic basket
Sterile nail (approximately 3 mm in diameter)

Procedure

Fruit disinfection
1. The fruits with uniformity and without physical injuries were used as experimental materials.
2. These fruits were immersed in 2% sodium hypochlorite solution for 2 min, rinsed with sterile tap water, and air-dried in a clean bench (approximately 2 h).
3. After being air dried, the fruits were wounded (approximately 4 mm in depth) at the equator with sterile nail (sterilized with alcohol lamp) prior to inoculation with pathogen (Figure 1).
  
  Figure 1. Wounding of apple fruit A and tomato fruit B with sterile nail
Pathogen inoculum preparation
1. Five microliter spore suspension (5 x 10⁶/ml in 15% glycerol, stored at -80 °C) of B. cinerea was inoculated on PDA plates and cultured for 2 weeks at 22 °C.
2. Fungal spores were harvested by flooding the surface of the culture with KH₂PO₄-glucose solution containing 0.05% (v/v) Tween-80, then filtered through four layers of sterile cheesecloth. The concentration of spore suspension was determined by observing under optical microscope using hemacytometer. Then, the concentration of spore suspension was adjusted to 5 x 10⁴ per milliliter by KH₂PO₄-Glucose solution.
Inoculation
Tomato and apple fruits were inoculated with 10 μl spore suspension (the suspension was mixed by Vortexer before inoculation) in each wounded site using a pipette, respectively, KH₂PO₄-Glucose solution was used as control. Ten inoculated fruits were put into plastic basket (40 cm x 30 cm x 10 cm) and sealed with plastic film, about 5mL sterile water was sprayed into the plastic film to maintain a high relative humidity (about 95%), and stored at 25 °C.
Disease scoring
Two days after inoculation, the lesion diameters were measured daily. Two diameter values of each lesion in two mutually perpendicular directions were recorded. The average of the two values was defined as the diameter of the lesion (Figure 2).

Figure 2. Lesions caused by B. cinerea and buffer. A. lesions on apple fruits (four days after inoculation). B. lesions on tomato fruits (3 days after inoculation). Buffer, KH₂PO₄-Glucose solution. (Source: Zhang et al., 2014)

Recipes

PDA medium (1 L)
Potato 200 g
Dextrose 20 g
Agar 15 g
Boiling 200 g of sliced potatoes in 1 L distilled water for 30 min, then decanting the broth through cheesecloth and adding 20 g dextrose and 15 g agar powder in the broth. Add distilled water to make up 1 L, and the medium is sterilized by autoclaving for 20 min.
2% sodium hypochlorite (5 L)
100 ml sodium hypochlorite
4.9 L dH₂O
KH₂PO₄-glucose solution (100 ml)
KH₂PO₄ 0.136 g (10 mM)
Glucose 0.198 g (10 mM)
Tween-80 50 μl (0.05%, v/v)

Acknowledgments

This protocol was adapted from Zhu et al. (2010) and Chan et al. (2007). The work was supported by the 973 program (grant number 2013CB127103), by National Natural Science Foundation of China (grant number 31030051) and by the Chinese Academy of Sciences (grant number KSCX2-EW-G-6).

References

Chan, Z., Qin, G., Xu, X., Li, B. and Tian, S. (2007). Proteome approach to characterize proteins induced by antagonist yeast and salicylic acid in peach fruit. J Proteome Res 6(5): 1677-1688.
Zhang, Z., Qin, G., Li, B. and Tian, S. (2014). Knocking out Bcsas1 in Botrytis cinerea impacts growth, development, and secretion of extracellular proteins, which decreases virulence. Mol Plant Microbe Interact 27(6): 590-600.
Zhu, Z., Zhang, Z., Qin, G. and Tian, S. (2010). Effects of brassinosteroids on postharvest disease and senescence of jujube fruit in storage. Postharvest Biol Technol 56(1): 50-55.