Observe the halo blight pathogen, Pseudomonas syringae pv. phaseolicola, in infected bean leaves and in culture.

Inoculate healthy bean seedlings with the halo blight pathogen and observe the development of symptoms.

Fulfill Koch's postulates using the P. syringae pv. phaseolicola-bean pathosystem.

One of the most common and economically important diseases of Phaseolus beans (snap beans, dry beans, etc.) grown in the Midwest is halo blight, caused by Pseudomonas syringae pv. phaseolicola. Disease development is favored by cool wet weather. The pathogen survives the winter in infected seed or plant debris and is disseminated primarily by windblown rain. P. syringae pv. phaseolicola can also be spread during cultivation, particularly when leaf surfaces are wet. Like most other bacterial leafspot pathogens, P. syringae pv. phaseolicola is capable of surviving as epiphytic populations on the surface of healthy soybean leaves. When these pathogenic bacteria enter the leaves through wounds or natural openings like stomates they multiply rapidly and induce the formation of lesions. The lesions, which are watersoaked at first, soon become brown and dry and are usually surrounded by a yellow halo. When many lesions grow together, large areas of dead leaf tissue may develop. The halo is actually caused by a toxin that is produced by the bacteria within the lesion. As the toxin diffuses out into the leaf tissue it causes breakdown of chlorophyll and creates the halo. When toxin from infected leaves is translocated to the growing point of the shoot, the new leaves that develop are often stunted and chlorotic.

P. syringae pv. phaseolicola grows on many common bacteriological culture media. Nutrient agar, plate count agar, and potato dextrose agar (all available from scientific supply houses such as Fisher, Sigma, Carolina Biological) all work very well. Some strains can lose virulence following repeated subculturing. For long-term storage, collect infected leaflets and allow them to air-dry for a few days at room temperature. Place dried infected tissue in small jars or ziploc bags and store in the freezer. To revive the bacteria, macerate some of the dried tissue in sterile water and streak onto plates of culture medium or use as inoculum to infect young bean seedlings as described below. I can usually provide cultures of this bacterial pathogen (on media or in infected dried leaves) to Iowa teachers for use in this exercise (contact ebraun@iastate.edu).

 

Bean varieties vary in their susceptibility to different strains of the halo blight pathogen. Each pathogen strain-bean variety combination should be tested to determine susceptibility. I have had consistent success with the bean variety Top Crop, but many other varieties are also susceptible to most strains of the pathogen.

 

Congo Red Staining Solution- 1 g congo red stain dissolved in 50 ml water.

 

Acid Alcohol- 3 drops conc. HCl in 30 ml 95% ethanol.

Bacterial Streaming- This is an important diagnostic technique that is used to determine whether pathogenic bacteria are associated with diseased tissue. Observe symptoms of halo blight on bean leaflets. Cut out a small piece of leaflet tissue containing one or more lesions. Make a cut directly through a lesion and place the tissue in a drop of water on a microscope slide. Apply a coverslip. Using the compound microscope, look for bacteria streaming out of the cut lesion. (Be sure to close the condenser aperture to increase contrast.) The bacteria should look like a brownish, granular mass oozing out from the infected tissue.

Bacterial Motility- P. syringae pv. phaseolicola is a motile bacterium. If slides prepared for observation of bacterial streaming are allowed to sit for 20-30 minutes, the bacteria can often be seen actively swimming in the fluid surrounding the leaf tissue. Motility is best observed at fairly high magnification (400X).

Congo Red Staining-

1. Place a drop of congo red solution on a clean slide and, using a clean toothpick, add a very small portion of a bacterial colony from a culture plate. Stir to distribute the bacteria well throughout the stain. Spread the stain in a thin film over the slide. (It is essential that the stain be spread very thinly over the surface of the slide.) OR To make a microscopic examination for bacteria in lesions on plants, place a lesion on a clean slide in a drop of the congo red solution. Chop the tissue in the stain with a razor blade and after a minute or so carefully scrape the tissue off the slide. The remaining stain is spread in a thin film over the slide.

2. Allow the stain to air dry.

3. Flood the dried film with acid alcohol to change the color of the dye to a deep blue, which is more distinct under the microscope.

4. Place a drop of immersion oil (or mineral oil) on the film and apply a coverslip. Observe at 400X. The bacteria stand out as white rods against a blue background.

Isolation of bacterial pathogens from plant tissues: Take a sample of symptomatic tissue and surface sterilize in 10% bleach for 15-30 seconds. Blot the tissue on a clean paper towel and place it in a tube containing 1 ml of sterile distilled water (use flamed forceps!). Now use a flamed glass rod and macerate the tissue in the sterile water. The lesion could also be macerated in a large drop of sterile water in a sterile petri dish. After a few minutes, transfer some of the bacterial suspension to culture media as follows:

a. Flame a transfer loop, let it cool, and pick up a loopful of the water containing the macerated plant tissue.

b. Streak the loop lightly over the surface of about one fourth of the plate to make many lines close together. Try not to let the lines overlap. (From this point on, the loop will NOT be returned to the suspension of macerated tissue.)

c. Flame and cool the loop again, draw it across the end of the previously streaked area once or twice, and streak about one-fourth more of the plate.

d. Repeat step c one or two more times.

e. Examine the plates in a few days. The colony type present in greatest frequency is quite likely that of the pathogen. Transfer cells from a single colony of typical morphology to new plate of culture media. Colonies of P. syringae pv. phaseolicola should be small and circular with a white to off-white color.

Inoculation Methods: The following are two common methods of inoculating plants with bacterial pathogens. Symptoms should be visible 7-10 days after inoculation, beginning as watersoaked lesions that look darker than the surrounding tissue with incident light and lighter than the surrounding tissue with transmitted light.

1. Wound inoculation: Prepare a faintly-cloudy suspension of P. syringae pv. phaseolicola in sterile water. Obtain a bean plant with leaflets that are 1/3 to 2/3 expanded. Fully-expanded leaflets are not susceptible. Now get some of the bacterial suspension on a piece of cheesecloth or cotton swab and rub the leaf surface while supporting the leaf from below with your other hand. Rubbing action will create small wounds and introduce the bacteria. Place plants on the greenhouse bench (or under grow lights or on a bright window sill).

2. Spray inoculation: Prepare a suspension of P. syringae pv. phaseolicola as in method 1 and place the bacterial suspension into a spray bottle. Spray a pot of bean seedlings to runoff with the bacterial suspension. Enclose the inoculated seedlings in a plastic bag for 24-48 hr. Remove the bag and place plants on the greenhouse bench.

Fulfilling Koch's Postulates: Use the techniques outlined above to 1) associate the bacterial pathogen with disease symptoms on infected plants; 2) isolate the pathogen and note its characteristics; 3) inoculate healthy soybeans with the isolated pathogen; 4) reisolate the pathogen from the inoculated plants and compare with initially isolated pathogen.

I can usually provide the microorganism Pseudomonas syringae pv. phaseolicola to Iowa teachers. Out of state teachers would have to get permits before I could send them this plant pathogen, even though it is common and not a risk. (Contact ebraun@iastate.edu).