Materials and Reagents

1.5 ml centrifuge tubes (Nanjing Qingke Biological Company)
50 ml centrifuge tubes (Nanjing Qingke Biological Company)
Long pot (20 cm tall x 5 cm diameter), hand-made with PVC tubes with a holed bottom cover
Parafilm (Bemis, catalog number: PM996 )
Press-in Saran Wrap (GLAD^R, Pressin Seal, 21.6 m x 30 cm), a type of sealing saran wrap with stickiness that can be pressed on the surface to seal tightly the object
Conical bottle (200 ml)
Paper towel (230 x 225 mm, Yong Li Yu Investment Company Limited, May Flower, catalog number: A18250S )
Cheese cloth (40S Warp x 40S Weft; 5 m x 1.2 m), made by combed cotton
Soil (Nanjing Shoude Company, nutrient soil: vermiculite = 2:1)
Syringe needle (Luer-Lok 20G, BD, catalog number: 309634 )
Plastic square trays (Purchased from market) (100 cm length x 80 cm width x 10 cm height)
Maize seeds (Inbred line: B73; select the similar size seeds with germinating rate > 90%)
Fungal strains: Fusarium graminearum (isolate: F0609); F. verticillioides (isolate: 7600)
Sterile ddH₂O
Glycerin
PDA (Potato dextrose agar) (Qingdao Hope Bio-Technology, catalog number: HB0233 )
Tween-20 (SunShine Bio, catalog number: T0014 )
Mung Bean (Purchased from market)
PDA media (see Recipes)
Mung bean soup (see Recipes)

Equipment

Pipettes (Eppendorf, Research Plus, catalog numbers: 3120000020 , 3120000046 , 3120000062 )
Hemacytometer (0.100 mm, Hausser Scientific, catalog number: 3110 )
Water bath (Changzhou Nuoji Instrument, catalog number: HHS-11-1 )
Clean bench (AIRTECH, model: SW-CJ-1FD , catalog number: A16116317)
Constant temperature incubator (Ningbo Jiangnan Instrument Factory, catalog number: DNP-9162 )
Growth chamber (Ningbo Jiangnan Instrument Factory, model: RXM-508C-3 , catalog number: 17091559)
Centrifuge (Eppendorf, model: 5424 , catalog number: 5424FL570190)
Microscope (Olympus, catalog number: BX41 )
Autoclave

Procedure

Growing maize Seedlings
1. Sow the seeds of different maize inbred lines in the soil in long pots made with PVC cylinders (5 cm in diameter and 20 cm in depth), with 4 seeds per cylinder, covered with a layer of soil in 2 cm depth. To prove the stalk rot phenotype, a resistant line 1145 and a susceptible line Y331 to Gibberella stalk rot, described by Yang et al. (2010) can be included as controls.
2. Place the pots in a controlled growth chamber (light cycle and temperature: 14 light/10 dark at 26 ± 2 °C; light intensity: 600 Lux) to allow the seedling growth.
3. Select the seedlings in similar size at approximately 12-d to 2-week-old stage and transfer to a culture room for infection assay.
Preparation of fungal spore suspension
1. Fungal strain culture
  Fungal isolates (F. graminearum or F. verticillioides) are initially stored in 20% glycerin at -80 °C. Inoculate the fungal isolate on a PDA plate and incubate it in an incubator (25 °C) in the dark for at least 10 days.
2. Stimulating sporulation
  Cut several agar blocks (5 x 5 mm) aseptically and place them in sterilized mung bean soup, and incubate in an incubator at 200 rpm, 28 °C for 2-3 d (Figure 1).
  
  Figure 1. Mung bean soup with F. graminearum cultured for 2 days
3. Spore enumeration and preparation of inoculum
  Filter the spore suspension in mung bean soup through two layers of cheese cloth, followed by centrifugation at 3,800 x g for 5 min. Discard the supernatant, re-suspend and wash the concentrated conidia twice with sterile water before diluting it to a concentration of 1.0 x 10⁶/ml in 0.001% Tween-20.
Inoculation of seedling stem with fungal spores
1. Preparation of maize seedlings
  Keep the plastic square tray (100 x 80 x 10 cm) in a controlled incubation room (light cycle and temperature: 14 light/10 dark at 24 ± 2 °C; light intensity: 200 Lux). To maintain a high humidity (~75%) inside the tray, add 250 ml sterile water to a layer of paper towel on the bottom of the tray, until the towel is wetted completely. Discard the extra water if needed. Line-up the pots with seedlings horizontally in the tray (Figure 2).
  
  Figure 2. The arrangement of pre-inoculation environment. Maize seeds are planted and grown in a PVC cylinder in a control room for 12-14 days. The pots with seedlings are then lined up horizontally in a tray with a layer of paper towel completely wetted on the bottom of the tray.
2. Artificial inoculation process
  To facilitate the infection of fungal spores, punch a tiny hole in approximately 1 mm depth in the middle position of first internode of the seedling stem using a syringe needle (Figure 3, Video 1), then drop 20 μl of spore suspensions in 0.001% Tween-20 to the wounded point carefully using a pipette (Figure 4, Video 2). Seal the plastic square tray with Press-in saran wrap to maintain the moisture. To allow the air exchange, make 6-8 uniform strip holes (in approximately 2 cm length) on the wrap. Upon infection, maintain the seedlings under a condition with 14 h light/10 h dark at 24 ± 2 °C for 3 d without moving (Figure 5). Check the humidity daily to ensure a high humidity at approximately 75%. Add the water if needed.
  
  Figure 3. Punching a hole on the stem of maize seedling
  
  Video 1. Punch a hole on the stem of maize seedling. Use a syringe needle to carefully make a tiny hole in approximately 1 mm depth on the first internode of maize seedling.
  
  Figure 4. Inoculation of maize stem with spore suspensions. 20 μl of spore suspensions in 0.001% Tween-20 is dropped to the wounded site on the seedling stem.
  
  Video 2. Inoculate maize seedling stem with Fusarium spp. spore suspensions. Drop 20 μl of spore suspensions in 0.001% Tween-20 to the wounded point carefully using a pipette.
  
  Figure 5. Maintainance of seedlings after inoculation. The plastic square tray is sealed with Press-in saran wrap to maintain a moisture > 75%. Several uniform holes are made on the cover surface. The tray is kept steadily in a controlled condition with 14 h light/10 h dark at 24 ± 2 °C.

Data analysis

Rating scales of disease severity
Score the stalk rot symptoms 3 days after artificial inoculation, with at least three scorings (15-20 seedlings per scoring per maize line). Based on the data collected from three or more scorings, estimate the disease severity for each individual plant using a 5-point classification scale ranging from 1 (highest resistance) to 5 (highest susceptibility) (Figure 6). Rating standards of corn stalk rot symptom are described in Table 1.

Figure 6. Phenotypic characteristics of seedlings by a 5-point classification scale. CK: non-inoculated control; 1, the most resistant; 5, the most susceptible.

Table 1. Scales and scoring standards of Gibberella stalk rot of maize seedling
Calculation of disease severity index (DSI)
Calculate the DSI according to the formula described by Ma et al. (2017) with some modifications:
DSI (%) = ∑(ranking value x number of plants with that value) x 100/(1 x total number of plants). All of the statistical analyses were performed using SPSS (14.0) software for ANOVA and the Student’s t-test, with the level of significance set at P < 0.05.

Notes

Select the healthy, non-stressed and uniform seedlings for infection assay.
If the inoculum drops immediately from the infection site after inoculation, redo the infection.
Maintain the appropriate humidity and temperature is the key for successful development and penetration of fungal pathogens.

Recipes

PDA media (200 ml)
9.2 g Potato glucose agar powder dissolved in 200 ml ddH₂O
Sterilize with autoclave for 15 min at 120 °C
Mung bean soup (1 L)
1. Boil 40 g Mung bean in 250 ml ddH₂O in a 85 °C water bath for 1 h
2. Then filter through a layer of gauze, and dilute in autoclaved ddH₂O to 1 L
3. Sterilize the soup at 120 °C for 15 min

Acknowledgments

This protocol is partially adapted from our previous work (Gao et al., 2007), with some modifications. This work was supported by The National Key Research and Development Program of China (No. 2016YFD0101002), the NSFC (No. 31471508, No. 31670702), and from the Technology Foundation for Selected Overseas Chinese Scholar, Ministry of Personnel of China (No. G0101500090) and the Innovation Team Program for Jiangsu Universities (2014). The authors declare no conflicts of interest or competing interests.

References

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