INTRODUCTION:

The Oomycetes, also known as water molds, are a large group of terrestrial and aquatic eukaryotic organisms. Although they superficially resemble fungi in mycelial growth and mode of nutrition, molecular studies and distinct morphological characteristics place them in the kingdom Chromalveolata (phylum Heterokontophyta, the 'stramenopiles') with brown and golden algae and diatoms.

Phylogenetic tree illustrating the approximate relationship between oomycetes and fungi (For further detail, see "Why are Phytophthora and other Oomycota not true Fungi?"

The terrestrial Oomycetes are primarily parasites of vascular plants, and include several very important plant pathogens. Aphanomyces, in the order Saprolegniales, causes a root rot of a wide range of hosts, including pea, snap bean, and sugar beet.

The order Peronosporales contains three families of plant pathogens. In the family Peronosporaceae, Plasmopara, Peronospora, Pseudoperonospora, Sclerospora, and Bremia are obligate parasites that cause serious foliar diseases known as downy mildews on many host plants such as grape, broccoli, onion, cucurbits, sorghum, and lettuce.

Broccoli leaf with signs of downy mildew, caused by Peronospora parasitica.(Courtesy G.A. Payne)

The family Pythiaceae contains obligate and nonobligate parasites, and includes the important pathogen genera Pythium and Phytophthora. Pythium species cause a variety of diseases including root rots of numerous plant species, Pythium blight of turf, and damping-off, which involves seed rot and pre- and post-emergence seedling death. Phytophthora species cause late blight of potato and tomato, foliar blights on peppers and cucurbits, and root or stem rots of many plant species. Phytophthora ramorum is a newly identified foliar pathogen that causes a disease called sudden oak death (also known as ramorum blight) on many trees and shrubs, and is a serious threat to forests and nurseries. Lessons on several diseases caused by oomycetes are available.

Damping-off of bedding plant seedlings due to Pythium. (Courtesy M.Daughtrey, copyright-free)	Conifer seedlings on the left are stunted and yellowed due to Phytophthora root rot.(Courtesy P.B. Hamm)
Symptoms and signs of late blight, caused by Phytophthora infestans, on a potato leaf. (Courtesy M. Powelson)	Advanced symptoms of sudden oak death in tanoak caused by Phytophthora ramorum (Courtesy E. Hansen)

The family Albuginaceae includes Albugo, an obligate parasite that produces a generally mild disease called white rust (not a true rust) on the stems, leaves, and fruit of cruciferous plants such as radish, horseradish, and several weed species.

White rust on leaf of cruciferous plant, caused by Albugo candida. (Courtesy P.A. Williams, copyright-free)

Although Oomycetes have previously been referred to as "lower fungi," they differ from fungi in several features. Oomycete cell walls contain cellulose, beta glucans, and the amino acid hydroxyproline, but do not contain chitin, which occurs in the cell walls of true fungi. The vegetative state of Oomycetes is diploid, whereas true fungi are haploid or dikaryotic. Oomycetes produce hyphae that are nonseptate (coenocytic), i.e. lacking in cross walls.

Asexual reproduction occurs in most Oomycetes by the formation of a structure called a sporangium (pl. sporangia) that arises on a specialized hypha termed a sporangiophore. Sporangia differ among various Oomycetes pathogens with respect to the shape of the sporangium, its mode of germination, the location of sporangia with respect to the host tissue, and the structure of the sporangiophores. In Aphanomyces, sporangia are filamentous and resemble vegetative hyphae. In Peronospora and other genera causing downy mildews, sporangiophores are branched and tree-like, with a single sporangium at the tip of each branch, and are often found emerging from the stomates.

Sporangia and sporangiophore of Peronospora parasitica, causal agent of downy mildew of cabbage. (Courtesy G.A. Payne)

Sporangial shape in Pythium ranges from filamentous in some species to round in other species. Sporangia of Phytophthora species are often lemon-shaped.

Lemon-shaped sporangia  of Phytophthora megasperma. (Courtesy F.A. Gray)

In Albugo, an unbranched sporangiophore produces a chain of sporangia. Clusters of sporangia beneath the plant epidermis form blisters that rupture as sporangia are released.

Sporangia of Albugo candida are produced on short sporangiophores beneath the epidermis of a mustard leaf. (Courtesy M.F. Brown and H.G. Brotzman)

Two mechanisms for germination of sporangia are known. The type of mechanism is often species-specific; however, some species can germinate by either mechanism depending on temperature or moisture conditions. In many of the foliar pathogens (e.g. Peronospora), sporangia germinate directly on the surface of a susceptible plant by the formation of a germ tube. A sporangium that germinates in this way is often referred to as a spore or conidium. Wind dispersion of sporangia of foliar pathogens is common.

Direct germination of a sporangium by germ tube in Phytophthora infestans. (Courtesy H.D. Thurston)

In most of the soil- and water-inhabiting genera, sporangia germinate indirectly by producing zoospores, which are asexual spores that are motile by means of two dissimilar flagella. The presence of a fibrous, anteriorly directed "tinsel" flagellum with hairs and a posteriorly directed "whiplash" flagellum also differentiates oomycetes from fungi, and supports their classification in the Kingdom Stramenopila.

In Aphanomyces and species of Phytophthora, differentiation of zoospores occurs in the sporangium. In contrast, in many Pythium species, the sporangium produces a short tube to which is connected a secondary structure referred to as a vesicle. Zoospores are partially differentiated in the sporangium, and transferred through the tube into the vesicle for final development and release.

	Zoospores being released from a sporangium of Phytophthora. (Used by permission of F.W. Schwenk, Kansas State University)
Zoospores are cleaved in sporangia borne at terminal ends of hyphae of Aphanomyces euteiches. (Courtesy M.F. Heimann, copyright-free)
Pythium undulatum, with round sporangia and thin vesicle surrounding zoospores just prior to release. (Courtesy P.B. Hamm)

Sporangia of some species are capable of germinating either by direct germ tube formation or indirectly by zoospore formation. The type of germination is influenced most strongly by temperature; for example, in Phytophthora infestans (the cause of late blight), cooler temperatures favor the formation of zoospores. For some soil-inhabiting species (e.g. Phytophthora cryptogea), the type of sporangial germination is also influenced by soil moisture; in general, very wet or saturated soil conditions induce zoospore formation, whereas slightly drier conditions favor direct germination.

Dispersion of zoospores occurs as they swim in water through soil pores or on the soil surface. They are often attracted by sugars and amino acids exuded by plant roots. Zoospores also can be dispersed passively in flowing water over longer distances, contaminating streams, canals, and ponds used for irrigation water. On susceptible plant surfaces, zoospores encyst and then germinate by producing a germ tube.

Sexual reproduction in Oomycetes occurs between two dissimilar gametangia: a large round oogonium containing one to several eggs, and a smaller antheridium that fertilizes the oogonium. If the antheridium is located at the side of the oogonium, the arrangement is termed paragynous. In an amphigynous arrangement, the oogonium grows through the antheridium, which remains as a collar at its base.

Paragynous arrangement of oogonium and antheridia in Pythium. (Courtesy P.B. Hamm) (arrows indicate antheridia)	Amphigynous arrangement of oogonium and antheridium in Phytophthora cambivora. (Courtesy P.B. Hamm) (arrows indicate antheridia)

In homothallic species, fertilization can occur in a single strain. In heterothallic species, two strains of opposite mating types are required for fertilization. In both homothallic and heterothallic species, fertilization results in a thick-walled zygote called an oospore. Oospores function as resting spores. They are produced in infected plant tissue, and are released into soil as the plant tissue degrades. They germinate either directly by a germ tube with or without a sporangium on the end, or indirectly by the formation of a vesicle with zoospores. As with sporangia, the type of germination is species and environment dependent.

Mature oospores of Aphanomyces within an infected root. (Courtesy Jennifer Parke)
	Disease cycle of Phytophthora cactorum. (Courtesy Jennifer Parke) Click image for an enlarged view.

MATERIALS

• Fresh specimens of downy mildew, late blight or other foliar blights, damping-off, and white rust, labeled with disease name and pathogen genus
• Grass leaves inoculated with Pythium spp.
• Dissecting microscopes
• Compound microscopes
• Razor blades
• Lactophenol cotton blue stain
• Clear Scotch tape (single- or double-sided)
• Microscope slides
• Coverslips
• Cultures and/or baits of Oomycetes showing sexual and asexual structures
• Prepared microscope slides (Plasmopara, Peronospora, Albugo)
• Pea or alfalfa seedlings
• Aqueous suspension of zoospores of Aphanomyces or Pythium

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PROCEDURES

A. Examination of fresh diseased material for symptoms of disease.

Examine disease specimens macroscopically. Record the disease name and pathogen genus on the following chart. Describe the symptoms you observe. Which physiological function(s) of the plant are most likely impaired by the disease?

B. Observation of fresh diseased material for signs of disease.

1. When foliar (disease) symptoms of late blight or downy mildew are present, examine diseased leaf tissue with a dissecting microscope to locate areas of sporulation (production of sporangia). Use the Scotch tape technique to make a spore mount. Touch the sticky side of a piece of clear Scotch tape to the sporulating area. Apply the tape, sticky-side down, to a drop of water on a microscope slide. Single-sided tape functions as its own coverslip; if double-stick tape is used, place a coverslip on top of it. Examine first under low, then under high power using a compound microscope. Observe the shape of the sporangia and their arrangement on the sporangiophore. Note the characteristic branching patterns of sporangiophores associated with different genera. Note also the nonseptate (coenocytic) hyphae typical of Oomycetes.

2. Use the lactophenol cotton blue stain to observe Pythium-infected grass leaves for oospores. With a razor blade slice thin sections of plant tissue from the advancing margin of a lesion. Place the tissue in a drop of lactophenol cotton blue on a microscope slide. Apply a coverslip, and examine first under low, then under high power using a compound microscope. Look for round, thick-walled oospores within the cells of the leaf tissue.

3. Record and diagram your observations on the chart.

C. Observation of reproductive structures in prepared cultures and slides.

1. Examine prepared cultures of Oomycetes under a dissecting microscope for oogonia, antheridia, and oospores. Note the location of the antheridium in relation to the oogonium. How do oospores differ from oogonia?

 2. Examine prepared slides using a compound microscope. Look for sporangia and sporangiophores.

a. Plasmopara viticola, cause of downy mildew of grape

b. Albugo candida, cause of white rust of crucifers

c. Peronospora parasitica, cause of downy mildew of broccoli

D. Observation of zoospores and their characteristics.

1. Zoospore motility: Sporangia in an aqueous preparation have been placed in water, chilled, and then warmed to induce the formation of motile zoospores. Observe the release of zoospores with a dissecting microscope or videoscope.

2. Attraction of zoospores to plant roots: Place the roots of a pea or alfalfa seedling into an aqueous suspension of Aphanomyces or Pythium zoospores in a petri dish, and let stand for 5-10 minutes. Observe the suspension with a dissecting microscope or videoscope for the accumulation of zoospores around the root zone of elongation.

 LAB SHEETS
[TO BE PRINTED AND FILLED OUT BY STUDENTS]