Protocol for diagnosing Erwinia amylovora infection using a fluorescent probe

Summary

Current fire blight diagnosis techniques are DNA based and require specialized equipment and expertise, or they are less sensitive. Here, we present a protocol for diagnosing fire blight using the fluorescent probe, B-1. We describe steps for Erwinia amylovora culture, implementing a fire blight-infected model, and E. amylovora visualization. This protocol allows for detection of fire blight bacteria of up to 10² CFU/mL on plants or objects in just 10 s with a simple application including spraying and swabbing.

For complete details on the use and execution of this protocol, please refer to Jung et al.¹

Graphical abstract

Highlights

• •Steps to establish a fire blight infection plant model
• •Details on a fluorescent probe for visualizing Erwinia amylovora infection
• •Application strategy of a fluorescent probe for the diagnosis of fire blight

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Current fire blight diagnosis techniques are DNA based and require specialized equipment and expertise, or they are less sensitive. Here, we present a protocol for diagnosing fire blight using the fluorescent probe, B-1. We describe steps for Erwinia amylovora culture, implementing a fire-blight-infected model, and E. amylovora visualization. This protocol allows for detection of fire blight bacteria of up to 10² CFU/mL on plants or objects in just 10 s with a simple application including spraying and swabbing.

Before you begin

Fire bright is a bacterial plant disease containing the pathogen Erwinia amylovora in which symptoms such as darkening, canker, and drooping appear in all plant organs.² As E. amylovora spreads rapidly through rain, wind, and insects, there is a considerable global economic burden for the control of fire blight.^3,4 Most fire blight diagnostic methods are based on DNA, which requires specialized equipment and experts and takes a substantial amount of time from sampling to analysis. Therefore, the development of a new diagnostic technology that can be used at the site of fire blight infection with rapid diagnosis and treatment is essential.

This protocol introduces newly developed fluorescence-based fire blight diagnostic techniques and the relevant procedures.

We recently reported the fluorescent probe B-1 that can visualize E. amylovora in plants or objects infected with fire blight.¹ B-1 emits negligible fluorescence with an excitation wavelength of 551 nm. However, the fluorescence intensity of B-1, especially at 686 nm, increases remarkably and emits red fluorescence under the presence of E. amylovora. There is great potential for applications with the various treatment strategies of B-1 toward the samples. We adopted spraying and swabbing as the simplest methods, which are described in detail in this protocol (Follow step 11–13 for spraying application and step 14–16 for swabbing application.)

Preparation of Erwinia amylovora

Timing: >12 h

• 1.
  Preparation of Luria-Bertani (LB) broth.

  • a.
    Dissolve 25 g/L LB broth cocktail in deionized water (DI H₂O) using a glass bottle. LB broth cocktail is replaceable with 10 g/L tryptone, 10 g/L NaCl, and 5 g/L yeast extract. Less than 10 mL of LB broth is required in this protocol.
  • b.
    Autoclave the solution at 121°C and 15 psi for 20 min.
  • c.
    Chill the LB broth at 25°C and store it at 4°C.

Note: If you want to make LB medium into an agar plate, add 15 g/L agar in step 1. After autoclaving them, pour 8–12 mL of the solution into a 100 mm petri dish.

• 2.
  Culture E. amylovora.

  • a.
    Take a few microliters of E. amylovora stock and add it to ∼5 mL of the LB broth (or spread it on the LB agar plate).
  • b.
    Incubate E. amylovora at 26°C for 12–16 h.⁵ It isn’t essential to incubate the bacteria in dark conditions but avoid direct sunlight.
  • c.
    Optional: agitating (150–400 rpm) could promote growth if the bacteria are cultured in LB broth.
• 3.
  Adjust the concentration of E. amylovora. Bacterial concentration adjustment can be calculated by spreading the bacteria on an agar plate.

  • a.
    Dilute the bacterial solution moderately.
  • b.
    Take 100 μL of diluted bacterial solution and spread to the LB agar plate using a sterilized spreader.
  • c.
    Incubate the LB agar plate at 26°C until the colonies appear. It takes more than 12 h but does not exceed a day which hinders accurate adjustment with large colonies.
  • d.
    Count the colonies and calculate the bacterial concentration with the dilution factor. 30–300 colonies per plate are suitable for exact adjustment. If the colony count is out of the range, re-dilute the bacterial solution to the appropriate concentration.

Note: Bacterial concentration adjustment can further be calculated with absorbance and Mcfarland turbidity. 0.1 in OD₆₀₀ and 0.55 in Mcfarland turbidity refers to 2 × 10⁸ colony-forming unit (CFU)/mL.¹

• 4.Optional: bacterial stock preparation

Bacterial stocks consist of pre-cultured bacteria solution and glycerol. Take 500 μL of the bacterial solution in step 2. b. into cryotube. Then add the same volume (500 μL) of autoclaved-50% glycerol solution (Additional mixing such as vortexing or pipetting isn’t required.) Label the bacterial strain and date, and store in a deep freezer (−80°C).

CRITICAL: Be cautious not to spill when handling E. amylovora as it spreads quickly and can pose an enormous risk to the plants. It is recommended to put a safety seat on the table. If there is a leakage of E. amylovora occurs, immediately report it to the relevant authority and follow their instructions.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Bacterial strain
Erwinia amylovora (TS3128)	Korea Research Institute of Bioscience and Biotechnology	N/A
Chemicals
BD Difco™ Dehydrated Culture Media: LB Broth, Miller	BD	Cat #DF0446-17-3
Ethyl alcohol, 99.5%	Samchun Pure Chemical	CAS #64-17-5
Ethyl acetate, 99.5%	Samchun Pure Chemical	CAS #141-78-6
n-Hexane	Samchun Pure Chemical	CAS #110-54-3
Sodiumhypochloritesolution	Samchun Pure Chemical	CAS #7681-52-9
Bromoacetonitrile	Sigma-Aldrich	CAS #590-17-0
Potassium carbonate	Alfa Aesar	CAS #584-08-07
N,N-Dimethylformamide	Alfa Aesar	CAS #68-12-2
Sodium sulfate	Samchun Pure Chemical	CAS #7757-82-6
Phenylboronic acid	Sigma-Aldrich	CAS #98-80-6
Trifluoroacetic acid	TCI	CAS #76-05-1
Palladium (II) trifluoroacetate	TCI	CAS #42196-31-6
6,6′-Dimethyl-2,2′-dipyridyl	TCI	CAS #4411-80-7
2-Methyltetrahydrofuran	ACROS Organics	CAS #96-47-9
Malononitrile	Sigma-Aldrich	CAS #109-77-3
Pyridine, anhydrous, 99.8%	Sigma-Aldrich	CAS #110-86-1
Silica gel 60 (0.040–0.063 mm)	Millipore	CAS #7631-86-9
Glass TLC plate, silica gel coated with fluorescent indicator F254	Supelco	Cat #1.05715.0001
Others
Heratherm™ Compact Microbiological Incubator	Thermo Scientific™	Cat #50125882
Spectrofluorometer	Shimadzu	Cat #RF-6000
Fluorescence Macro-cuvettes Quartz Glass	Hellma	Cat #101-10-40
Petri dish	SPL Life sciences	Cat #10090
15 mL conical tube	SPL Life sciences	Cat #50015
UV flashlight	Alonefire	Cat #SV004
Software
ImageJ	National Institutes of Health	https://imagej.nih.gov/ij/download.html

Step-by-step method details

The procedure for the establishment of fire blight-infected plant model, the synthesis of B-1, and its application are described below. In the treatment strategy of B-1 to detect fire blight bacteria, follow step 11–13 for spraying and step 14–16 for swabbing.

Establishment of the fire blight-infected plant model

Timing: > 24 h

The protocol below describes how to establish a fire blight-infected plant model in detail with readily available plants, for example, apple trees. If available, a naturally infected sample may be used instead of an experimental model.

• 1.Collect the plant tissues such as blossom, leaf, and stem using scissors or pruning shears in the desired size. A size of under 50 × 50 mm is recommended for incubation in experimental conditions.
• 2.Briefly sterilize the plant tissue by dipping it in 70% EtOH or 0.1% NaClO solution for 30 s and wipe off using tissue paper.

Optional: Performing this step in a sterile hood can avoid further contamination, but it isn’t essential.

• 3.Soak the whole plant tissue or the petiole of the leaf in the LB broth with 10⁸ CFU/mL of E. amylovora using a culture dish or conical tube (Figure 1).
• 4.Incubate the plant tissue for 24 h at 26°C (Shaking isn’t required in this step.) Some black blots, one of the symptoms of fire blight, will appear on the plant tissue, but it may differ depending on the plant species and organs (Figure 1).

Note: Another strategy is to use a brush for the fire blight-infected plant model. Dilute the desired concentration of bacteria (CFU/mL) with a solvent such as DI H₂O. Then, apply the bacteria solution twice or more on the surface of the plant tissue using a brush sterilized by autoclaving or 70% EtOH. This can imitate the case in which only a few E. amylovora exists on the surface of plants in the early stage of fire blight.

Figure 1.

Establishing the fire blight-infected plant model

(A) Schematic diagram of establishing the fire blight-infected plant model.

(B) Apple tree leaves incubated in a petri dish containing LB broth without (left) or with E. amylovora (right).

(C) Apple branch with blossoms and leaves infected with fire blight by incubating in petri dish containing LB broth.

(D) An apple tree leaf infected with fire blight by dipping the petiole of the leaf and the magnified images showing the symptoms of fire blight.

Synthesis of B-1

Timing: > 3 days

The protocol below describes the synthetic procedure of B-1. The synthesis of B-1 consists of 3 steps and is based on core structure (DMHNA) which was prepared according to the previously reported synthetic route (Figure 2).⁶ Organic solvents and silica are used in all synthetic steps, it must be performed in the hood. For the information of NMR and Mass spectroscopy of each compound, please refer to Jung et al.¹

• 5.
  Synthesis of Compound 1.

  • a.
    Flame dry the 10 mL round bottom flask with the stirring bar.
  • b.
    Add a mixture of DMHNA, bromoacetonitrile, potassium carbonate, and N,N-dimethylformamide (DMF) in the flame-dried round bottom flask.

    Reagent	Molecular weight	Mole	Equivalent	Amount
    DMHNA	215.25 g/mol	0.139 mmol	1 eq	30 mg
    Bromoacetonitrile	119.95 g/mol	0.209 mmol	1.4 eq	14 μL
    Potassium carbonate	138.205 g/mol	0.209 mmol	1.4 eq	29 mg
    DMF	73.09 g/mol			5 mL
    Total				5 mL

  • c.
    Stir the reaction and heat up at 50°C for 2 h under argon atmosphere.
  • d.
    Work up the reaction. Chill the reaction at 25°C and dilute with 50 mL of ethyl acetate (EtOAc). Using 250 mL funnel, wash the reaction mixture with 50 mL of DI H₂O 3 times and 50 mL of brine once.
  • e.
    Separate the organic phase into a 250 mL conical flask and add 5 g of sodium sulfate. Stir the mixture for 20 min for eliminating the remaining water.
  • f.
    Extract the organic phase by filtration and evaporate. In the filtration, further washing step helps to increase the yield of the reaction.
  • g.
    Purify the reaction mixture by silica column chromatography. If EtOAc/Hexane (Hex) (3:7, v/v) is used as eluent, the yellow fluorescent substance corresponding to Rf 0.25 on thin layer chromatography (TLC) is the product.
  • h.
    Dry the solvent and confirm using NMR and Mass spectroscopy.
• 6.
  Synthesis of B-C.

  • a.
    Flame dry the 10 mL Schlenk tube with the stirring bar.
  • b.
    Add a mixture of Compound 1, phenylboronic acid, trifluoroacetic acid, palladium (II) trifluoroacetate, 6,6′-dimethyl-2,2′-dipyridyl, and 2-methyltetrahydrofuran in the flame-dried Schlenk tube.

    Reagent	Molecular weight	Mole	Equivalent	Amount
    Compound 1	254.28 g/mol	0.115 mmol	1 eq	29 mg
    Phenylboronic acid	121.93 g/mol	0.230 mmol	2 eq	28 mg
    Trifluoroacetic acid	114.02 g/mol	1.15 mmol	10 eq	88 μL
    Palladium (II) trifluoroacetate	332.45 g/mol	0.09 mmol	5 mol%	30 mg
    6,6′-Dimethyl-2,2′-dipyridyl	184.24 g/mol	0.18 mmol	10 mol%	33 mg
    2-Methyltetrahydrofuran	86.13 g/mol			1.5 mL
    Total				1.6 mL

  • c.
    Stir the reaction and heat up at 80°C for 24 h under argon atmosphere.
  • d.
    Follow the work up protocol above (Step 5. d-f).
  • e.
    Purify the reaction mixture by silica column chromatography. If EtOAc/Hex (1:9, v/v) is used as eluent, the orange fluorescent substance corresponding to Rf 0.25 on TLC is the product.
  • f.
    Dry the solvent and confirm using NMR and Mass spectroscopy.
• 7.Synthesis of B-1.
• a.Flame dry the 10 mL 2-neck round bottom flask with the stirring bar.
• b.
  Add a mixture of B-C, malononitrile, and pyridine in the flame-dried round bottom flask.

  Reagent	Molecular weight	Mole	Equivalent	Amount
  B-C	315.37 g/mol	0.032 mmol	1 eq	10 mg
  Malononitrile	66.06 g/mol	0.063 mmol	2 eq	4 mg
  Pyridine	79.10 g/mol			1.5 mL
  Total				1.5 mL

• c.Stir the reaction and connect one of the necks to the reflux condenser. Heat up the reaction at 70°C for 16 h under argon atmosphere.
• d.Follow the work up protocol above (Step 5. d-f).
• e.Purify the reaction mixture by silica column chromatography. If EtOAc/Hex (1:4, v/v) is used as eluent, the purple substance corresponding to Rf 0.2 on TLC is the product.
• f.
  Dry the solvent and confirm using NMR and Mass spectroscopy.

  Pause point: The synthetic step can be paused after each step (5–7). Evaporate the solvent from the purified compound and store it at -20°C.

Figure 2.

Synthetic scheme of B-1

The yield of each step is Compound 1; 92%, B-C; 41%, and B-1; 41%.

Detection of E. amylovora in the laboratory

Timing: <10 min

In the experimental setting, E. amylovora can be detected using a spectrofluorometer. When diagnosing the fire blight with the fluorescence spectrum, even minute differences can be identified that are difficult to detect with the naked eye.

• 8.Rinse the samples with DI H₂O. The volume may differ by the size of samples but is recommended fewer for distinct detection.
• 9.Add 10 mM of B-1 stock up to 10 μM into the rinsed solution.
• 10.Measure the fluorescence spectrum using a spectrofluorometer (RF-6000, Shimadzu) at an excitation and emission wavelength of 551/686 nm (Figure 3).

Note: It is recommended to store B-1 in the DMSO stock solution (10 mM) at -20°C. To detect the extremely low concentration of E. amylovora (10² CFU/mL), use a concentration of B-1 less than 10 μM. Although the fluorescent intensity of B-1 is extremely low, it may interfere with the interpretation of fluorescence change by E. amylovora.

Figure 3.

Detecting E. amylovora using a spectrofluorometer

(A) The emission spectrum of 10 μM B-1 with 0–4.3 × 10⁸ CFU/mL of E. amylovora in DI H₂O. Excitation: 551 nm.

(B) Emission intensity plot of (A) at 686 nm. The emission spectrum and intensity were obtained using RF-6000 (Shimadzu) without further calculation.

Detection of E. amylovora by spraying B-1

Timing: <10 min

The following protocol describes how to detect E. amylovora on the surface of plants or objects by spraying the fluorescent probe (B-1). The spraying method can be generally applied to any sample materials for detecting E. amylovora.

• 11.Dilute B-1 with DI H₂O to produce a 100 μM solution.
• 12.Spray the B-1 solution 2–3 times over the samples.
• 13.Check the fluorescence change using a UV flashlight (Figure 4).

Note: It is recommended to conduct this procedure under dark conditions to enhance the clarity of the fluorescence image. If the samples emit fluorescence or have strong auto-fluorescence, the fluorescence change of B-1 might not be clearly distinguished.

Figure 4.

B-1 application method: spraying

(A) Schematic diagram of detecting E. amylovora on plants and farming tools by spraying B-1.

(B and C) (B) An apple blossom, (C) pruning shear, and trowel after spraying 100 μM of B-1 3 times. The pictures were taken using a 10 W 365 nm flashlight.

Detection of E. amylovora by swabbing

Timing: <10 min

Chlorophyll in plants emits a red fluorescence under UV, which can disturb the diagnosis of fire blight by the fluorescence change of B-1.⁷ The following swabbing method protocol can mitigate the fluorescence interference of chlorophyll.

• 14.Soak the cotton swab in the 100 μM B-1 solution.
• 15.Rub the samples with the cotton swab soaked in B-1.
• 16.Check the fluorescence change of the swab using a UV flashlight (Figure 5).

Note: It is recommended to conduct this procedure under dark conditions to enhance the clarity of the fluorescence image.

Figure 5.

B-1 application method: swabbing

(A) Schematic diagram of detecting E. amylovora on plants by swabbing B-1.

(B) The B-1-soaked swab (100 μM) after rubbing the fire blight-infected apple leaf. The pictures were taken using a 10 W 365 nm flashlight.

Expected outcomes

B-1 is a turn-on type fluorophore that emits red fluorescence in the presence of lipopolysaccharide of Erwinia amylovora. Spectrofluorometer-based fire blight diagnosis demonstrates that the increment of the peak of 686 nm is observed in a bacterial concentration-dependent manner (Figure 3A). The exact concentration of bacteria in the samples can be further determined with a standard curve (Figure 3B). In general uses, the B-1 treatment on plants or objects that are presumed to be infected by fire blight shows a red fluorescence in the area only where E. amylovora is present (Figures 4B, 4C, and 5B). The spread of fire blight can be efficiently controlled by removing the identified infected area or treating it with antibiotics.

Limitations

This protocol describes a new fluorescence-based platform for diagnosing fire blight. The fluorescent probe for detecting Erwinia amylovora, B-1, can detect low concentrations of the fire blight pathogen within a few seconds. However, there are some limitations to overcome. During the daytime in the orchards, it can be difficult to clearly identify fluorescence changes due to the sunlight. Since this fire blight diagnosis platform with B-1 relies on a red fluorescence, plant tissues containing chlorophyll can be mistakenly diagnosed as fire blight. However, this can be overcome through additional applications, such as swabbing. To enable the extensive use of B-1, further studies, and research shall be pursued on its effects on plants or animals and how the related problems can be solved.

Troubleshooting

Problem 1

When performing silica column chromatography after synthesis, the product cannot be found or only the side products are shown in TLC (Related to Synthesis of B-1).

Potential solution

Depending on the experimental environment, the synthesis may not proceed correctly. When reacting, dry the flask completely and make sure to proceed with the reaction under an argon atmosphere. It may be helpful to collect some of the reactants during the reaction and check the TLC.

Problem 2

It is difficult to identify the fluorescence change of B-1 outdoors (Related to Step 13 and Step 16).

Potential solution

We recommend moving the sample to the shade and observing it using a UV flashlight with an output of 10 W or higher.

Problem 3

It is difficult to distinguish the fluorescence of B-1 from the auto-fluorescence of plant tissue or objects (Related to Step 13).

Potential solution

Chlorophyll is present in plant organs, especially in leaves. It has a wide range of emissions from red to far-red with a peak at 683 nm, making it difficult to distinguish it from B-1. We recommend the swabbing method to occlude the auto-fluorescence of the sample. Swabbing is free from fluorescence interference as it only collects E. amylovora present on the surface of the sample. (See detection of E. amylovora by swabbing section.)

Problem 4

The fluorescence change cannot be observed in the fire blight-infected samples (Related to Step 13 and Step 16).

Potential solution

When the concentration of E. amylovora on the surface of the sample is extremely low, it may be difficult to see the fluorescence change with the naked eye. We recommend taking pictures before and after treating B-1 under the UV light and analyzing them using an image processing software such as ImageJ. In ImageJ, the brightness of the fluorescence can be measured by Analyze—Measure tool.

Problem 5

After treating the sample with B-1, orange fluorescence is observed, not red. (Related to Step 13 and Step 16).

Potential solution

If the surface area of the sample is sharp and narrow, a blue shift in fluorescence may occur. In addition, due to the self-fluorescence of the bacteria, some samples with high concentration of bacteria show orange fluorescence.

Resource availability

Lead contact

Further information or requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dokyoung Kim (dkim@khu.ac.kr).

Materials availability

Where available, these may be shared by the lead contact.

Data and code availability

This study did not generate datasets or code.