II. THE FLAGELLATE FUNGI

Today's laboratory exercise will focus on fungi that produce zoospores at a certain stage of their life cycle. Because of this characteristic, they are also often referred to as zoosporic fungi. This group contains some of the more interesting and perhaps most destructive fungi in terms of pathogenicity on plants and animals. Unlike the slime molds, the flagellate fungi have cell walls composed of chitin, cellulose, or ß-1,3 glucans; and they obtain their nutrients by absorption. In addition, they possess coenocytic hyphae and reproduce via zoospores. They are mostly water or soil inhabiting organisms. The following phyla will be considered in this laboratory:

Chytridiomycota - characterized by posteriorly uniflagellate zoospores
Hyphochytriomycota - possess anterior uniflagellate zoospores
Oomycota - all produce motile biflagellate zoospores or planogametes

I. PHYLUM HYPHOCHYTRIOMYCOTA

This phylum has only one order, Hypochytridiales, and includes those organisms that produce zoospores with tinsel-type anterior flagella. It consists of a small number of species that occur in soil, freshwater, and marine habitats. Some members are hyperparasites of Oomycota and the zygospores of endomycorrhizal fungi and may control populations of these fungi. No specimen is available from this phylum for today's exercise.

II. PHYLUM OOMYCOTA

This is a large, important group of zoosporic fungi that have biflagellate zoospores, one flagellum being of the tinsel type and the other a whiplash. Some members of this phylum are among the most destructive known and have influenced world events. The Great Potato Famine of 1845 was the result of an epidemic case of late blight of potato in Ireland. The causal agent is Phytophthora infestans. The Oomycota have been moved from the Kingdom Fungi to Stramenopila due to some odd characteristics.

A. ORDER SAPROLEGNIALES

This order is considered the most primitive of the Oomycetes. Most members occur in freshwater habitats or soil and are saprophytic; though a few are parasitic on higher plants, fungi and animals, including fish and their eggs. Many members, particularly Saprolegnia spp., have large diameter hyphae (200 µm); whereas the more advanced group of Oomycetes are characterized by fine hyphae. Most of the Saprolegniales have several oospores per oogonium which is considered more primitive than having one oospore per oogonium. Sporangium morphology and zoospore release are used in separating genera. The life cycle of Saprolegnia is shown in Fig. 6.

Observe Saprolegnia and Achlya growing on hemp seeds. Draw and record your observations.

B. ORDER LEPTOMITALES

This group is separated from the Saprolegniales because they have hyphae that are constricted and plugged with cellulin. Their sporangia tend to be less hyphal than those of the Saprolegniales. Oogonia in this group usually have a single oospore which is considered a more advanced characteristic.

C. ORDER LAGENIDIALES

This order of Oomycota has uncertain affinities. They are parasitic on algae, other zoosporic fungi, higher plants, or animals. The thalli of this group are usually holocarpic and monocentric with the entire sporangium being converted to zoospores that are discharged into a vesicle that is usually outside the host. Some are animal parasites in the marine habitat; and Lagenidium giganteum, a freshwater member and mosquito parasite, is of economic importance.

D. ORDER PERONOSPORALES

The Peronosporales are the most specialized of the Oomycota. It includes aquatic, amphibious, and terrestrial species; and it culminates in a group of highly specialized obligate parasites. There are three families in this order: Pythiaceae, Peronosporaceae, and Albuginaceae.

1. FAMILY PYTHIACEAE

Members of the Pythiaceae occur in aquatic, intermediate, or terrestrial habitats. A few of the terrestrial species cause significant damage to economic plants as represented by the genera Pythium and Phytophthora. These fungi are either saprophytes or facultative parasites that are easily grown on artificial media.

a. Observe the oogonium, antheridium and oospore of P. aphanidermatum and Pytophthora capsici. Note the similarities and differences of these structures in each.

b. Observe vesicle formation in Pythium dissotocum and release of zoospores, also in P. aphanidermatum and P. capsici (`cat in a sack' release characteristics . Obtain a plate of with sporangia ready to release zoospores and observe under the dissecting scope ( or the compound scope at 40X or 100X). Record your observations.

c. Observe zoospore chemotaxis. Zoospores of pathogenic Pythium spp. are attracted to certain chemicals produced by the roots of plants (root exudates) they will infect. Obtain a plate containing active zoospores of P. aphanidermatum and place it under the microscope. Check the activity of the zoospores. Do not move them too much or they will stop swimming. Place the root of a plant into the water and within a good microscopic field of view. Observe the reaction of the zoospores. Record your observations.

d. How fast can Pythium aphanidermatum kill a host plant? What are the requirements for successful disease development? In this set of experiments, you will try to answer these questions. To be done with a partner. Obtain four plates of healthy cucumber seedlings. Inoculate one pot with P. aphanidermatum by placing mycelial and sporangial agar plugs onto the media. Label your inoculated plates. The other two plates will be the uninoculated control seedlings. Incubate one set of inoculated and control pot at 30 °C and incubate the other set at 25 °C. Observe and record your observations at 1, 2 and 3 days after inoculation.

2. FAMILY PERONOSPORACEAE (The Downy Mildews)

This family together with the next one, are considered the most specialized of the Oomycota. They are all obligate parasites and are differentiated from each other by their sporangiophore characteristics. See Fig. 8 for the distinguishing sporangiophores of the different genera.

a. Observe a unique downy mildew affecting the root of lettuce. Into what genus would you place this downy mildew?

b. Observe other available slides of Plasmopara viticola - downy mildew of grape, and Peronospora tabacini - blue mold of tobacco.

3. FAMILY ALBUGINACEAE

This family is commonly known as the 'white rusts' organisms. Members of this family produce sporangia in chains on club-shaped sporangiophores in sori developing under epidermis of hosts. Important species include Albugo candida which attacks brassicas, A. ipomoeae-panduranae which attacks sweet potato and morning glory, and A. occidentalis which attacks spinach.

Observe prepared slides of Albugo sporangia and sporangiospores. Draw and record your observations.

III. PHYLUM CHYTRIDIOMYCOTA

All zoospore producing fungi that have a single posterior whiplash flagellum are included in this phylum. It is believed that there are two evolutionary lines in this phylum, one line includes the orders Spizellomycetales and Blastocladiales, the other the Chytridiales and Monoblepharidales. Thallus types range from monocentric, holocarpic forms to polycentric, rhizomycelial forms to truly mycelial forms. The sexual reproductive processes in these fungi also vary and are accomplished in one of three ways:

Isogamy - fusion of morphologically identical gametes (eg. Olpidium)
Anisogamy - fusion of morphologically different gametes (eg.Allomyces)
Oogamy - fertilization of a non-motile female gamete by a motile male gamete (eg. Monoblepharis)

A. ORDER CHYTRIDIALES

This order consists of organisms commonly referred to as chytrids. Their usual habitat is freshwater, although there are a few that inhabit soil and marine environments. A number of these organisms are parasitic on fungi, algae, and higher plants. No mycelial forms occur in this order, and some members have limited rhizoidal development. Most are saprophytic and thus have little economic importance, but there are two economically important plant parasites:Synchytrium endobioticum is the causal organism of potato wart and Olpidium brassicae infects the root of cabbage and other plants.

The life cycle of Olpidium viciae and Synchytrium endobioticum are illustrated in Figs. 1 and 2. Observe prepared slides of host cells infected with these chytrids. Record your observations.

B. ORDER BLASTOCLADIALES

The members of this order occur in moist soils and freshwater habitats. They may be parasitic on animals or on plants. Saprophytic members may be found on decaying animal or plant parts, including fruits and twigs. Some members are wholly fermentative and carboxyphilic in their metabolism, producing large quantities of lactic acid. Physoderma spp.(eg. P. zeae-maydis, causal organism of brown spot of corn) and Coelomomyces spp. (mosquito parasites) are obligate parasites and have not been cultured apart from their hosts. Coelomomyces is unique among zoosporic fungi in that the gamete-bearing and spore-bearing phases of the life cycle occur on two different hosts. The diploid thallus of members of this order characteristically produces thick-walled, melanized resting structures that are the site of meiosis. Zoospores of this order have a nuclear cap, a distinctive internal structure which represents a more highly developed form of the zoospore with compartmentalization.

The life cycle of these fungi are very similar to those found in the algae. The life cycle of three interesting genera, Coelomomyces, Allomyces and Blastocladiella are shown in Figs. 3, 4, and 5.

Observe prepared slides of Allomyces and be familiar with the different structures. Draw what you see under the microscope.

C. ORDER MONOBLEPHARIDALES

The Monoblepharidales are ecologically more like the Chytridiales in that they are primarily aquatic. No members of this group are important plant or animal parasites. However, their unique method of sexual reproduction is of interest among researchers. Three genera make up this order: Monoblepharis, Monoblepharella, and Gonapodya.