Materials and Reagents

Protection gloves (Medicom, catalog number: 1174D )
Protection coat (Medicom, catalog number: 8018 )
Cellophane membrane (DINGGUO CHANGSHENG, catalog number: XH444-1 )
Round (90 mm diameter) Petri dishes (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 101VIRR )
Sterilized gauze (Thermo Fisher Scientific, Fisher Scientific, catalog number: 22-415-469 )
Pot for cotton growth (Shantou Xing Lv Yuan, dimensions: 300 x 200 x 100)
Sterilized pipette tips (Corning, Axygen^®, catalog number: TF-1005-WB-L-R-S )
50 ml conical tubes (Corning, catalog number: 430828 )
Cotton seeds (Nongqishi Agricultural Institute, cv. Xinluzao NO.16)
Potato tubers
Glycerol
Distilled water (Milli-Q) (EMD Millipore, catalog number: QTUM00ICP )
70% (v/v) ethanol (EtOH in ddH₂O)
30% H₂O₂ (ALADDIN, catalog number: H112519 )
Glucose (Sigma-Aldrich, catalog number: V900392 )
Agar (BD, Bacto^TM, catalog number: 214010 )
Sodium nitrate, NaNO₃ (EMD Millipore, catalog number: 106537 )
Potassium dihydrogen phosphate, KH₂PO₄ (EMD Millipore, catalog number: 104873 )
Potassium phosphate dibasic, K₂HPO₄ (Sigma-Aldrich, catalog number: V900050 )
Magnesium sulfate heptahydrate, MgSO₄·7H₂O (EMD Millipore, catalog number: 105886 )
Potassium chloride, KCl (EMD Millipore, catalog number: 104933 )
Sodium citrate (Sigma-Aldrich, catalog number: V900095 )
Zinc sulfate heptahydrate, ZnSO₄·7H₂O (Sigma-Aldrich, catalog number: Z4750 )
Iron(II) sulfate heptahydrate, FeSO4·7H2O (Sigma-Aldrich, catalog number: V900038 )
Ammonium iron(III) sulfate dodecahydrate, NH₄Fe(SO₄)₂·12H₂O (Sigma-Aldrich, catalog number: V900032 )
Copper(II) sulfate pentahydrate, CuSO₄·5H₂O (Sigma-Aldrich, catalog number: C7631 )
Manganese(II) sulfate monohydrate, MnSO₄·H₂O (Sigma-Aldrich, catalog number: V900271 )
Boric acid, H₃BO₃ (Sigma-Aldrich, catalog number: V900267 )
Sodium molybdate dehydrate, Na₂MoO₄·2H₂O (EMD Millipore, catalog number: 106521 )
Sucrose (Merck Millipore, catalog number: 107687 )
Murashige and Skoog (MS) medium including vitamins (Duchefa Biochemie, catalog number: M0222 )
Potato dextrose agar (PDA) (see Recipes)
TES (see Recipes)
Minimal medium (MM) (see Recipes)
MS liquid medium (see Recipes)
Czapek-Dox medium (see Recipes)

Equipment

Heat plate and cooking pot to boil potatoes (Supor, catalog number: SDHC8E15 )
Flasks and magnetic stirrer for preparation of solutions (Thermo Fisher Scientific, Fisher Scientific, catalog number: S88850206 )
Fungal growth incubator (SAIFU, catalog number: MJX-450S )
26 °C incubator shaker (Zhicheng, catalog number: ZHWY-2102C )
Autoclave (Hirayama, catalog number: HVE-50 )
Clean bench (Zhicheng, catalog number: ZHJH-C1209B )
Hemocytometer for conidia counting (Qiujing, catalog number: QJ1102 )
Rotator (Qilinbeier, catalog number: QB-208 )

Procedure

Penetration assays
1. Pipette 20 μl conidial suspension (3 x 10⁷ conidia per ml) of V. dahliae stored at -80 °C in 20% glycerol and plate the suspension culture onto the PDA plate and incubate for 5 days at 26 °C in the dark.
2. Prepare Minimal medium (MM medium) and autoclave 20 min at 113 °C. Pour plates after cooling the medium down to 50-60 °C and dry the plates in a clean bench.
3. Cut the flat sheet membranes of cellophane into circles of 90 mm diameter and autoclave 20 min at 113 °C. Lay one circle on each MM plate prepared in step A2.
4. Use a sterilized pipette tip to inoculate the fungi from step A1 onto the cellophane membrane overlaid on the MM medium. Grow cultures for 3 days at 26 °C in the dark. Then remove the membrane and incubate the medium for 7 days to observe hyphae in the underlying medium for determination of breach of cellophane (Figure 1).
  
  Figure 1. Penetration assay on the cellophane membrane. A. Uncovering the membrane from the MM plates with a colony of V. dahliae at 3 days post inoculation (dpi). B. The colony of V. dahliae on the underlying medium indicating the penetration of cellophane membrane.
Pathogenicity assays
Susceptible upland cotton (cv. Xinluzao NO. 16) and strain V592 of V. dahliae were used in our lab for pathogenicity assays.
1. Sterilize cotton seeds for 15 min and rinse three times with distilled water. Soak the seeds overnight at room temperature. Germinate seeds in pots filled with wet soil and cover with plastic dome for one week to grow seedlings until two cotyledons are fully expanded.
2. Transplant 12 seedlings per pot with MS liquid medium and grow at 26 °C with a 16 h light (8,000 lux)/8 h dark cycle for about 2 weeks before inoculation of V592.
3. Streak V592 onto fresh PDA plates (from older PDA plates or glycerol stocks) and incubate for 1 week. Transfer one plate (90 mm) of mycelia by a sterilized blade into 200 ml Czapek-Dox medium with shaking at 200 rpm for 3-4 days at 26 °C in the dark to obtain conidia.
4. Prepare the conidial suspension through filtrating the fungal cultures with 4 layers of sterilized gauze to remove mycelia. Measure the concentration of conidia in the suspension using a hemocytometer and adjust to approximately 1 x 10⁷ conidia per ml with Czapek-Dox medium.
5. Immerse the roots of seedlings with two true leaves to the conidial suspension from step B4 for 1 h (Figure 2A). Put back the inoculated seedlings to MS liquid medium and supply water once a week.
6. Count disease incidence by the percentage of cottons that showed wilting symptom at 30 dpi (Figure 2B). The infection assay was repeated at least three times with 3 pots of cotton each trial.
  
  Figure 2. Pathogenicity assay of V. dahliae. A. Root-dip inoculation of cotton seedling with conidia suspension. B. Representative disease symptom in cotton at 30 dpi of V. dahliae.
Fungal recovery
To identify the colonization of V. dahliae in vascular tissues, we isolate the inoculated cotton stems to recover the fungi.
1. Cut the stem above cotyledons of cotton at 30 days post inoculation (Figure 3).
2. Sterilize the stem sections for 1 min in 70% ethanol within a 50 ml conical tube followed by 60 min in 40 ml of 10% (v/v) H₂O₂ (dilute the 30% [v/v] H₂O₂ with distilled water). Revolve the tube on the rotator at 20 rpm during this process.
3. Rinse the stems three times with sterilized water for 3 min each time and culture at 26 °C on PDA medium in the dark. The colonies of V. dahliae can grow on the stems within 1 week (Figure 3).
  
  Figure 3. Recovery of V. dahliae from the infected stem of cotton

Data analysis

Data from at least three biological repeats were obtained and one-way ANOVA was used to analyze the disease incidence with Microsoft Excel (Table 1).

Table 1. Analysis of disease incidence. Disease incidence was counted by the percentage of diseased cottons in 3 pots of infected cottons (3 x 12, total 36 cottons). Assay was repeated 3 times.

Notes

Each of cellophane membranes should be separated by a filter paper to prevent adhesion during autoclaving.
To prevent shrinkage of the cellophane membrane, it is soaked into sterilized water firstly before putting on MM plate.

Recipes

Potato dextrose agar (PDA)
Boil 200 g of peeled potatoes in 1 L of distilled water for 20 min
Filtrate through 4 layers of gauze
Add 20 g of glucose and 20 g of agar
Distilled water to 1 L
Sterilize by autoclaving 20 min at 113 °C
TES
50 g/L citrate
50 g/L ZnSO₄·7H₂O
50 g/L FeSO₄·7H₂O
13 g/L NH4Fe(SO4)₂·12H₂O
2.5 g/L CuSO₄·5H₂O
0.5 g/L MnSO₄·H₂O
0.5 g/L H₃BO₃
0.5 g/L Na₂MoO₄·2H₂O
Stored at 4 °C
Minimal medium
2 g/L glucose
2 g/L NaNO₃
1 g/L KH₂PO₄
0.5 g/L MgSO₄·7H₂O
0.5 g/L KCl
0.02% (v/v) TES
20 g/L agar
Sterilize by autoclaving 20 min at 113 °C
MS liquid medium
0.1% (w/v) MS
Czapek-Dox medium
30 g/L sucrose
3 g/L NaNO₃
1 g/L K₂HPO₄
0.5 g/L MgSO₄·7H₂O
0.5 g/L KCl
100 mg/L FeSO₄·7H₂O
Sterilize by autoclaving 20 min at 113 °C

Acknowledgments

This protocol was developed from the following published paper: Bourett and Howard, 1990. This work was supported by grant from the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB11040500) and the China Transgenic Research and Commercialization Key Special Project (2014ZX0800908B).

References

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