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​​Instructor Notes​

Fresh diseased plant material.

Available materials will vary according to location and season. Some suggestions for diseased plants follow. Place specimens in a plastic box lined with moist paper toweling, cover with a lid, and store in a cold room at 5ºC. Specimens can be successfully stored for up to 3-4 days.

a. Late blight of tomato: symptoms and signs may be found in home gardens on both the foliage and the ripening fruit. Lesions with sporangia are most easily found in the early morning when the relative humidity is high and temperatures are cool, conditions favoring reproduction of the pathogen.

b. Phytophthora blight of pepper and cucurbits: on pepper, look for circular gray-brown water-soaked lesions on leaves, black stem lesions, and lesions on fruit with mycelium and often sporangia. Typical manifestation on cucurbits is a fruit or stem rot.

c. Damping-off: Plant some vegetable seeds such as pea into flats containing garden soil. Keep cool and wet after emergence to promote damping-off.

d. Grass leaves with Pythium: Plant grass seeds in pots and inoculate with a Pythium species such as P. aphanidermatum or P. ultimum, which are common causal agents of foliar blights. Cover with plastic bags and keep wet at 28-30ºC. Foliar blighting, mycelium, and oospores will be produced in 5-7 days.

Downy mildew occurs on emerging onions and peas in the spring, and on mature plants in the fall if the weather has been cool and wet. Infection of broccoli, cucurbits, and grapes results in foliar lesions that are chlorotic on the upper leaf surface and whitish on the underside. Examine the underside of the leaf with a dissecting microscope for sporangia and sporangiophores, which are often visible during damp weather or early in the morning when dew is present.

White rust is frequently found on weeds such as pigweed (Amaranthus), morning glory (Ipomoea), or shepherds purse (Capsella bursa-pastoris) in the fall of the year. Diseased plants can be found along roadsides or along the borders of recently plowed fields.

Prepared specimens

Prepared slides of asexual and sexual reproductive structures of Plasmopara viticola, Albugo species, Phytophthora infestans, and Peronospora species can be purchased from Triarch, P.O. Box 98, Ripon, WI 54971.

1. Suggested isolates

a. Some species that will produce sporangia in culture are Phytophthora cactorum, P. capsici, P. nicotianae, and P. palmivora.

b. Homothallic species for production of oogonia, antheridia, and oospores in culture include most species of Pythium. Homothallic species of Phytophthora with paragynous antheridia are P. cactorum, P. citricola, and P. megasperma. Homothallic species of Phytophthora with amphigynous antheridia are P. erythroseptica, P. fragariae, P. heveae, and P. boehmeriae.

c. Recommended species for the exercise "Attraction of zoospores to plant roots" are Pythium aphanidermatum (NRRL #29623) and Aphanomyces euteiches (NRRL # 29642). They may be obtained from the ARS Culture Collection.

2. Sources for isolates: Isolates of Oomycetes can be obtained from the following culture collections: Mycologists and plant pathologists at land grant universities

• Mycologists and plant pathologists at land grant universities

• ARS Culture Collection (NRRL Culture Collection)
  National Center for Agricultural Utilization Research (NCAUR)
  Agricultural Research Service, U.S. Department of Agriculture
  1815 N. University St.
  Peoria, IL 61604
  (309) 681-6383
  http://nrrl.ncaur.usda.gov/

• American Type Culture Collection (ATCC)
  10801 University Blvd.
  Manassas, VA 20110-2209
  (703) 365-2700
  http://www.atcc.org  

  Note: A USDA Animal and Plant Health Inspection Service, Plant Protection and Quarantine permit (no. 526) will be required to obtain some of the pathogens listed from another state (including the culture collections).

3. Baiting techniques: A number of techniques have been developed for isolating Phytophthora and Pythium species from soil and water (refer to Basic Plant Pathology Methods, Dhingra, O.D. and Sinclair, J.B., CRC Press, 1995, second edition). Isolates obtained this way may form reproductive structures on the baits. For culturing isolates on artificial media for development of sexual and asexual spores, identification to genus and species may be necessary, as methods vary from species to species.

a. Baiting soil for Pythium

Sprinkle a thin (2-3 mm) layer of any field soil onto a paper towel and spray with water to moisten. Place 20 untreated corn or pea seeds on the soil. Roll the paper towel into a cylinder, place it inside a plastic bag, and incubate at room temperature. After 4-5 days, examine the seeds for fluffy white mycelium emerging. To demonstrate the importance of Pythium as a seed rot and damping-off pathogen, the procedure can be performed with both untreated seeds and seeds treated with the fungicides metalaxyl or captan.

b. Baiting soil for Phytophthora

Some foliage baits for Phytophthora and their targeted species are: apple fruits or cotyledons for P. cactorum, P. cinnamomi, and P. citricola; lupine seedlings for P. megasperma, P. nicotianae, P. cinnamomi, P. parasitica, and P. syringae; eucalyptus cotyledons for P. cinnamomi; strawberry leaves or roots for P. fragariae; alfalfa seedlings for P. megasperma; sunflower seedlings for P. cactorum; and Port Orford-cedar foliage for P. lateralis.

A general baiting method is to place infested soil approximately 1 cm deep in a watertight container such as a cottage cheese carton, or a styrofoam cup for small soil samples. Add distilled or deionized water to cover the soil to a depth of about 2 cm. (If the soil contains a large amount of organic matter that might float, the double cup baiting method can be used. Place the soil in the bottom of a styrofoam cup. Cut out the bottom of a second cup, and cover the cutout area with a layer of cheesecloth. Nest the bottomless cup into the sample and add water.) Allow mud to settle, then float the bait on the water and incubate at room temperature under fluorescent light for 2 days to 1 week. With many species of Phytophthora, a fringe of sporangia will accumulate around the bait. To culture, transfer the entire bait or discolored infected portions to corn meal agar or to a selective medium such as PARC agar (recipe below), and incubate at room temperature. Phytophthora will grow on the agar in 2 to 3 days.

Phytophthora cactorum can be isolated from most apple orchard soils using apple cotyledons. Place air-dried soil into a watertight container, moisten the soil, and incubate for 4 days at room temperature. Then flood the soil with deionized water, chill at 4-6 C for 2 hours, return to room temperature, and float the cotyledons on the water. Sporangia will form on the fringe of discolored tissues after several days.

c. Baiting water sources for Phytophthora

Bartlett pears, green apples such as Granny Smith, and other fruits can be used as baits. Use fruit that is firm, free of wounds, and reasonably free of blemishes to minimize non-target infections and aid recognition of infection spots. Place single fruits in open mesh bags and float them in the water source. Protection from predators may be necessary. Leave bags in the water for several days to 1 to 2 weeks, depending on convenience, propagule density, and competition for the baits. Remove the fruits from the bags, wash and dry them, and place them in a plastic box lined with paper towels and covered with a lid. Incubate at room temperature for up to 1 week. Examine the fruits at intervals for surface brown spots indicative of internal decay; fruits may be split open through the decayed areas. The texture of Phytophthora decay is usually firm, distinguishing it from that caused by Penicillium or other saprophytes. Transfer small portions of discolored tissue to corn meal agar, or preferably to a selective medium such as PARC agar (recipe below).

d. Baiting water sources with popcorn baits

Obtain water from a relatively clean natural source such as pond or stream, and place in an uncovered jar. Place several popped popcorn kernels in the water, and allow to stand for several days. Mycelia of Oomycete organisms will appear in 2-4 days, with asexual sporangia and zoospores at about the same time. Oogonia and antheridia will appear a few days later, beginning on the oldest mycelium. To provide a range of stages of reproductive growth, start jars consecutively 3, 4, and 5 days prior to the lab session. Click here for a K-12 exercise that describes this technique.

4. Protocols: Some general methods are given below. Detailed protocols for the handling of particular genera and species may be found in Methods for Research on Soilborne Phytopathogenic Fungi, (ed. by Singleton, L.L., Mihail, J.D., and Rush, C.M., APS Press, 1992). This includes chapters on Pythium, Phytophthora, and Aphanomyces, with recipes for culture media in the Appendix. Additional protocols for induction of asexual reproduction in Pythium and Phytophthora species are provided in Tables 15 and 16 of Plant Pathological Methods, Fungi and Bacteria (John Tuite, Burgess Publishing Company, 1969).

a. Media: Aphanomyces and Pythium species grow rapidly on corn meal agar. Pythium and Phtytophthora spp. grow well on V-8 juice cholesterol agar; the presence of exogenous sterols often is necessary for production of reproductive structures. Selective media for recovery of Oomycetes baited with plant tissue include PARC agar (corn meal agar amended with ampicillin, rifampin, and pimaricin) for Phytophthora and Pythium, and Metalaxyl-Benomyl-Vancomycin (MBV) agar for Aphanomyces. Although incubation temperatures vary, a range of 22-26 C is satisfactory for many organisms.

b. Sporangia production: Cultures of Phytophthora and Pythium species can often be stimulated to form sporangia by the addition of water, either to an agar culture or by washing and resuspending the mycelial mat in a broth culture. Repeated rinsing with water or mineral salts may be necessary to deplete nutrients in the culture. Some species require light for the production of sporangia; others require an exogenous source of sterols. Optimal incubation temperatures also differ among genera and species (see detailed protocols in the references listed above). Sporangia may be observed at the margin of the colony.

c. Zoospore release: For many species, sporangia can be stimulated to release zoospores by adding a small amount of water to a sporulating culture, and first chilling the culture, then placing it at room temperature. The duration of chilling required ranges from 15 min to 2 hours; specific temperatures vary according to the species used.

d. Attraction of zoospores to plant roots: To produce zoospore suspensions of Aphanomyces euteiches NRRL # 29642, grow the organism for 3-5 days on corn meal agar. Place 5 mm disks of the culture in a petri dish, and flood with mineral salts solution. Primary zoospores will be produced in 6-24 hours, and secondary zoospores will be released from the primary spores 4-8 hours later. To use Pythium aphanidermatum NRRL #29623, grow the organism on corn meal agar. Transfer several agar plugs from an actively growing culture to 25 ml of V-8 Juice cholesterol broth, and incubate for 24 hours at 24 C. Rinse mycelial mats with sterile distilled water and incubate overnight. Rinse 3 times with mineral salts solution at 1-hour intervals. Zoospores will be produced 5 hours after the last rinse.

Germinate alfalfa seeds in a jar; pea seeds may be germinated in moist paper towels. For the experiment, place the roots or the whole pea or alfalfa seedling in a petri dish containing the suspension of zoospores. Within 5-10 minutes, the zoospores will accumulate around the zone of elongation on the roots.

e. Sexual structures: Oogonia, antheridia, and oospores of Aphanomyces and Pythium are readily formed after several days' growth on corn meal agar. Pythium and Phytophthora will produce sexual structures on V-8 Juice cholesterol agar. Phytophthora megasperma and P. cactorum form oospores quickly and abundantly in single culture.

Culture media and strains

Corn Meal Agar

• 17 g Difco corn meal agar

• 1 liter distilled water

• Boil to dissolve completely.

• Autoclave 15 min at 121ºC.

V-8 Juice Agar

• 200 ml V-8 juice

• 2.5 g CaCO₃

• Combine and centrifuge to clarify (5 min at 2,000 rpm). Decant and discard pellet.

Add:

• 20 g agar

• Distilled water to make 1 liter

• Autoclave 15 min at 121ºC.

V-8 Juice Cholesterol Broth or Agar (from Methods for Research on Soilborne Phytopathogenic Fungi)

• 200 ml V-8 juice

• 2.5 g CaCO₃

• Combine and centrifuge to clarify (30 min at 13,200 g).

Add:

• 2 ml of cholesterol (30 microgram/ml) from a 1.5% solution (2 mls) in 95% ethanol.

• (Add 15 g agar for solid medium.)

• Autoclave 15 min at 121ºC.

PARC Agar (Phytophthora Selective Medium) (from Methods for Research on Soilborne Phytopathogenic Fungi)

• 17g Difco corn meal agar

• 1 liter distilled water.

• Autoclave 20 min at 121ºC. Cool to 45ºC.

Add:

• 10 mg pimaricin (20 mg of 50% a.i.)

• 10 mg rifampicin (100% a.i.)

• 250 mg ampicillin (100% a.i.)

• Protect from light during preparation, storage, and incubation of media.

MBV Agar (from Methods for Research on Soilborne Phytopathogenic Fungi)

• 10 g Difco Bacto agar

• 10 g Difco corn meal agar

• 1 liter distilled water

• Autoclave. Cool to 50ºC and add:

• 30 mg metalaxyl (E.C. dissolved in 95% ethanol at 10 mg a.i.)

• 5 mg benomyl (wettable powder)

• 200 mg vancomycin

• 0.5 mg amphotericin B (optional for suppression of Alternaria, Rhizopus, Mucor)

Mitchell and Yang Salts Solution for zoospore production (from Methods for Research on Soilborne Phytopathogenic Fungi)

• 192 mg CaCl₂   

• 74 mg KCl

• 246 mg MgSO₄.7H₂O

• 1 liter distilled water

• Adjust pH to 6.5. Autoclave.

Lactophenol cotton blue stain (phenol may be omitted to make lactoglycerine cotton blue stain)

• 20 g phenol crystals (dissolve by gentle heating)

• 20 ml lactic acid

• 40 ml glycerol

• 20 ml distilled H₂0

• 0.05 g cotton blue (aniline blue)

Lab sheets

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