The fungi: Myxomycota and Oomycota

The fungi: Myxomycota and Oomycota

Introduction

We will begin this section of the class with the fungi in the phyla Myxomycota and Oomycota. These two phyla are now known to be distantly related from each other and also from the Fungi. The Myxomycota are commonly referred to as the plasmodial slime molds are classified in the Super Group Amoebozoa. The Oomycota are referred to as water molds and are classified in the Kingdom Stramenopile, the same as in the algal phyla Bacillariophyta, Chrysophyta and Phaeophyta. The Super Group in which they are classified has varied, but has most recently been classified in the Chromalveolata or SAR (Acronym for Kingdoms Stramenopile, Alveolata and Rhizarians that make up this Super Group). The Stramenopile is believed to form a monophyletic line. As we cover these two phyla, you will be able to see that they do not have the combination of characteristics of that defines the Kingdom Fungi and will see why they are no longer classified with the true Fungi.

 Phylum: Myxomycota (Plasmodial Slime Molds)

There are approximately 900 species of organisms that are classified in the Amoeboza super group. Of these, there are three groups of organisms that are classified as slime molds that are not closely related to one another. These groups include cellular slime molds, net slime molds and plasmodial slime molds. Of these, we will only consider the latter group.

There are approximately 850 species of plasmodial slime molds. They are found on moist soil, decaying wood, and dung. One of the interesting characteristic about this group of organisms is in their distribution. While species of other organisms will vary in different geographical localities, i.e. you don't find the same species of plants and animals on the mainland that you find in Hawai‘i, this is not generally true of the Myxomycota. Most species can be found throughout the world.

Plasmodial slime molds are so called because of its plasmodium (pl.=plasmodia) assimilative stage. This stage typically consists of a large, slimy, fan-shaped structure that is composed of branched tubules of protoplasm that feeds mostly on bacteria, but is also known to consume other microorganisms and plant and animal debris. This stage lacks a cell wall, and as it grows, the diploid nuclei divide, synchronously, by mitosis without cytokinesis. Thus, the plasmodium is essentially a large, single-celled, naked mass of protoplasm. Although the plasmodium can be observed to feed on particulate, organic material in nature, it is also able to grow, in vitro, on dissolved organic material, on an agar medium by absorbing nutrients in the agar. The plasmodium ingest particulate food material as it migrates by extending itself around the food particle. Once the protoplasm has enclosed the material, a cell membrane, the food vacuole separates it from the rest of the protoplasm and hydrolytic enzymes are released that will digest the food. Waste material remains in the vacuole and is later released from the plasmodium. The process by which this type of ingestion  occurs is called phagocytosis. A second, haploid assimilative stage, the amoeba (pl.=amoebae), also occurs in the life cycle of plasmodial slime molds. Its characteristic differs mostly in size from the plasmodium stage. It differs mostly in being microscopic and unicellular with a single nucleus. Like its giant counter part, it also ingest food via the process of phagocytosis. This process is very different from absorption, the mode of nutrition observed in Fungi. Other characteristics that are absent is the lack of cell wall in the assimilative stage and lack of mycelium and yeast stages. The Myxomycota were originally classified as fungi because they reproduce by spores that are produced in sporangia. This classification was made at a time when fungi were more broadly defined.

Although the amoeba stage is common to all amoebozoa, this is not an indicator of relationship. The amoeba stage has evolved several times and can also be found in other super groups, as well.

The life cycle of Myxomycota will vary with species, but most research has been carried out with Physarum polycephalum and will be used as representatives of the Myxomycota.

Life Cycle of Myxomycetes

Spore Germination, Amoebae and Swarm Cells

The spores of Myxomycetes are haploid and are normally globose and uninucelate. The spore surface may range from almost smooth to spiny. Spores of P. polycephalum are spiny (Fig. 1). The spore wall is composed primarily of cellulose and is only one of two stages where a cell wall is formed. The other stage that forms a cell wall is the microcyst, which is discussed below. Upon germination, the spore will crack open and release a single, uninucelate amoeba (Fig. 2). Amoebae will increase in numbers, asexually, as they feed and reproduce by mitosis and cell division.

The amoeba stage may continue to proliferate for an indefinite period of time if there is available nutrient and the environment remains favorable. The amoeba stage may also vary according to the environment. When free water is available, the amoeba is able to convert itself into flagellated swarm cells (Fig 4-5), a form more appropriate for submerged in water. In a dryer environment, the amoeba form is present. During periods of unfavorable conditions, the protoplast of the amoeba or swarm cell can round up and form a thin, cellulose  protective layer around itself, called the microcyst (Fig 6), which will protect it from the harsh environment. When favorable conditions return, the amoeba stage will emerge from the microcyst.

 

Figure 1: Physarum polycephalum spore.
Figure 2: Physarum polycephalum spore germination.


Figure 3: A pair of amoebae, under phase contrast. This stage may serve as an assimilative stage, as well as for gametes during sexual reproduction. Figure 4: Swarm cell with one flagellum visible, under phase microscopy. When immersed in water, amoeba transforms into swarm cell.

Figure 5: Several swarm cells, under phase microscopy. Flagella not visible.

Figure 6: Resistant microcyst stage forms during unfavorable conditions.

Zygote and Plasmodium Formation

After a period of time, when a critical number of swarm cells or amoebae have formed, sexual reproduction will occur and these assimilative stages will then also function as gametes. In P. polycephalum, it is the swarm cells that normally act as gametes. However, amoebae derived the same population, are usually self sterile. In order for syngamy to occur, gametes must be derived from a different population of amoebae, and be of a different mating strain. By convention, different mating strains are designated as a1, a2, a3, etc. and syngamy will occur when amoebae of different mating strains come together, e.g., a1 X a2, a1 X a3, etc. Once syngamy occurs the zygote is formed. The zygote undergoes numerous mitotic divisions to form the large, multinucleate plasmodium. Plasmodial slime molds are also commonly referred to as acellular slime molds because the plasmodium (Figs. 7-8) is essentially a large single, multinucleate cell. As is the case in the amoebae stage, the plasmodium is also an assimilative stage that consumes food by phagocytosis.  The appearance of the plasmodium is variable. In  P. polycephalum (Fig. 7), it is bright yellow, while in Didymium iridis (Fig 8), it is colorless.

During periods of unfavorable conditions for the plasmodium, a protective, brittle layer around itself and becomes dormant. This dormant stage is termed a sclerotium (Fig. 9), and if observed under the microscope, it can be observed to be composed of a number of smaller multinucleate units called macrocysts (Fig 10). Upon return of favorable conditions, each macrocyst can give rise to a new plasmodium.

Figure 7: Bright yellow plasmodium of P. polycephalum.  Figure 8: Colorless plasmodium of D. iridis. 
Figure 9: The plasmodium becomes dormant, forming a sclerotium when conditions become unfavorable. Figure 10: Section through sclerotium showing it is actually composed of smaller units called macrocysts. 

Formation of Sporangia

During favorable conditions, the plasmodium continues to feed and grow. However, when food and/or water becomes limiting, formation of sporangia will occur. A sporangium is a structure in which spores are borne and stored until their dispersal. Light appears to be another stimulus to sporangia formation in plasmodial slime molds. 

The P. polycephalum sporangium is a dark gray to almost black, lobed sporangium that is produced on a yellowish stipe (Fig. 9). The fragile, outer layer of the sporangium that encloses the spores is the peridium (pl.=peridia), which may be persistent or degenerate by the time the sporangium is ready to disperse its spores.

When sporangium formation begins, the outer portion of the plasmodium becomes denser, forming a thick called the hypothallus (Fig. 11). The protoplasm of the plasmodium then becomes divided into discrete units (Fig. 12), that will each give rise to a sporangium. Each unit will initially elongates (Fig. 13), and as development continues, the basal portion will decrease in diameter, to become the stalk, while the upper portion becomes the sporangium proper and will develop the finger-like projection characteristic of P. polycephalum (Fig.14). 

Upon the completion of movement of protoplasm into the sporangium proper, the stalk has now become more constricted and is devoid of protoplasm. Spore formation comes about with the formation of cell walls around the diploid nuclei. The nucleus in each spore will undergo meiosis to produce four 1N nuclei. Of these, three will degenerate. Thus, each diploid nucleus results in the development of only a single spore. Also, throughout the interior of the sporangium is the branched, thread-like capillitium. The capillitium arises from coalescence of vacuoles, which contain various material from the protoplasm, including calcium carbonate (CaCO3) in some species.

Figure 9: Sporangia of P. polycephalum are lobed, dark gray to black that are borne on stalks.

Figure. 10: A L - section through a strand of plasmodium with a thick hypothallus. Figure 11: L - section showing plasmodium divided into discrete units.
Figure 12: L - section showing pressure exerted by protoplasm to give sporangium its characteristic shape.  Figure 13: L - section of now mature sporangium. Stalk now devoid of protoplasm, with nuclei now restricted to sporangium.

Sporangium Variations in Myxomycetes

We have just described sporangial development of P. polycephalum, a species that is stalked with a lobed sporangium and persistent peridium . However, sporangia of plasmodial slime molds are very variable.

Most sporangia are small, ranging from less than 1 to several millimeters and sporangia may be stalked (Fig. 14)  or sessile (Fig. 15) and are borne in clusters from a single plasmodium. The number of sporangia and sporangial density are variable, but usually consistent with species. However, some species produce much larger sporangia. The largest type of sporangium is an aethalium (Figs. 16-17). It is usually described as cushion-shaped and in the case of Brefeldia maxima, may be as large as 1 m2 and weigh as much as 44 pounds. The large size of aethalia are presumed to be due to the fusion of tightly clustered sporangia that have completely fused. Sterile threads present in aethalia have a different origin than those of capillitium of other plasmodial slime molds and are referred to as pseudocapillitium. Their origin is from the common peridial walls of the fused sporangia that make up the aethalium. There are species that have tightly clustered sporangia that form a cushion-shaped mass that resemble aethalia, but individual sporangia remain distinct during development. These clusters are referred to as a pseudoaethalium (Fig. 18). The last type of sporangium is the plasmodiocarp (Fig. 19). It is a sessile sporangium that takes the shape of the veins of the plasmodium from which it is derived and for that reason can be as large as the plasmodium.  

Figure 14. Diachea leucopodia is another species with stalked sporangia. Peridia are breaking apart relvealing the capillitium, beneath. Figure 15. Trichia favoginea is an example of a species that produces sessile sporangia. Inset shows several  tightly clustered sporangia.
Figure 16. Lycogala epidendrum is an example of a species that produce aethalia. This species is probably one of the most common slime molds. Figure 17. The aethalium of Fuligo septica may grow to be more than 28 inches across, making it one of the largest slime mold.

Figure 18. Tubifera ferruginosa is an example of a pseudoaethalium. They may be confused with an aethalium because of their tightly clustered sporangia. Picture from:
http://www.hiddenforest.co.nz/slime/family /reticulariaceae/retic01.htm

Figure 19, Hemitrichia serpula is an example of a species that produces plasmodiocarps. The veins of the plasmodium are entirely converted  into a single sporangium and retains the shape of the plasmodium.

Variations in Capillitium Morphology

Capillitium (pl.=capillitia) are filamentous structures that develop with the spores within sporangia. They are thought to function in the retention of spores in the sporangia. By retaining spores the capillitium will allow gradual dispersal of spores over a long period of time. Thus, if over a period of time there are occasions when dispersal of spores will be unfavorable for amoebal and plasmodial growth, some spores will always be retained that will be dispersed at a later, possibly during more favorable conditions. Capillitia are often ornamented and have been used in defining some taxa in Myxomycetes. Some variations are illustrated below (Figs. 20-23):

Figure 20. Capillitium and spores of Trichia favoginea. Capillitium has a spiral, rope-like ornamentation. Picture from: http://www.photomacrography.net/forum /userpix/96_sporescapillitumCZweb_1.jpg
Figure 21. Capillitium of Hemitrichia serpula differ from most species in that they are unbranched, occurring in distinct strands called elaters. Ornamentations have a spiral, rope-like appearance.
Figure 22. Pseudocapillitium and spores of Lycogala epidendrum. The origin of of pseudocapillitium differs from capillitium. They are interpreted as remnants of the peridial walls of the fused individual sporangia that make up the aethalium. Figure 23. Capillitium of Stemonitis axifera form a network that originates from the columella, the extension of the stalk into the sporangium. Picture from: http://www.hiddenforest.co.nz/slime/family /stemonitidaceae/images/stemo05n.jpg



Phylum: Oomycota (Water Molds)

The name Oomycota literally means "egg fungus". The Oomycota are currently classified in the Kingdom Stramenopila. They have also been classified in the Kingdom Chromista as well, but this is only a name change. The Oomycota were also once classified as fungi, but unlike the slime molds, many species are fungal in their characteristics and appearance. For example, many species produce extensive mycelium that grow in their food by deriving their nutrition by absorption. However, they are no longer classified as fungi because of various non-fungal characteristics such as cell walls that are composed of cellulose rather than chitin, their life cycle being gametic rather than zygotic and their biflagellate zoospores, with one whiplash flagellum and one tinsel type flagellum, i.e. they are heterokonts. Flagellated Fungi, i.e., Phylum Chytridiomycota, have a single whiplash flagellum attached to the posterior end of the cells of their gametes and zoospores. Their mitochondria also differ from Fungi. In cross section, the inner membrane of mitochondria, the cristae, are interconnected by small diameter tubular structures called tubular cristae (Fig. 24). Fungi have the typical convulated, inner membrane that form platelike cristae (Fig 25).


Figure 24: Tubular cristae of mitochondria, typical of Oomycota. Figure 25: Platelike cristae of mitochondria,typical of Fungi

The Oomycota, are thought to be most closely related to brown algae (Phaeophyta) and diatoms (Bacillariopohyta), and form a monophyletic line with these groups even though the Oomycota are not photosynthetic. Characteristics that they share include cell walls composed of cellulosezoospores that are heterokonts, and mycolaminarin as a storage reserve, a compound similar to that of the laminarin present in brown algae and diatoms.

The origin of this group is believed to have been driven by endosymbiosis. Following the origin of the chloroplast that resulted from primary endosymbiosis, with the engulfment of a Cyanobacterium ancestor, there was a divergent into two lines: One leading to the Green Algae and the other to the Red Algae. Each line was later involved in secondary endosymbiosis, the further engulfments of unicellular ancestors by heterotrophs that evolved into further protistan, photosynthetic lines. One of the lines derived from a unicellular red alga ancestor gave rise to the Stramenopila line (Fig. 26). Although, as indicated above, the Oomycota are not photosynthetic, has this always been true? In many taxa that have photosynthetic ancestors, vestigial remnants of chloroplasts may be found in their cells (Tyler, et al. 2006). For example, Plasmodium, the genus of organisms that cause malaria, has a non-functional, vestigial chloroplast that is referred to as an apicoplast. However, no such structure has been observed in the Oomycota. Nevertheless, sequencing of the genome of two species of Phytophthora, P. sojae Kaufm. & Gerd. and P. ramorum Werres, De Cock & Man has determined that there are numerous genes of photosynthetic origin, which supports the hypothesis that the Stramenopile ancestor was photosynthetic (Tyler, et al. 2006).

Figure 26: Primary and secondary endosymbiosis as a driving force of evolution of photosynthetic protista. Image from Campbell, Introduction to Biology, 8th. edition.

We will examine two representatives in the Oomycota: Saprolegnia sp., in the order Saprolegniales and Phytophthora palmivora, in the order Peronosporales. The former life cycle will be is used as representative of the Oomycota.






Saprolegnia (Saprolegniales):

Asexual Reproduction: Asexual reproduction occurs with the formation of flagellated spores, zoospores, that are borne in zoosporangia. There are two types of zoospores that may form in the Saprolegniales. The primary zoospore (Fig. 27) that is pear shaped and has its flagella inserted in posterior end of the cell. This stage will swim for a period of time and will round up and encyst, becoming dormant for a period of time.  A morphologically different zoospore will later emerge from the cyst that is bean shaped and laterally flagellated (Fig. 27)

In Saprolegnia, zoosporangia are not morphologically differentiated from mycelium and resemble hyphal cells. However, they can be easily be located since they are delimited by septa and have much denser protoplasm than the rest of the mycelium. Following release of zoospores, Saprolegnia zoosporangia can produce more zoosporangia by the internal proliferation of a new zoosporangium inside of the previous one (Figs. 28-29). This is one characteristics by which genera may be defined. For example, compare Saprolegnia with the genus Achyla, which as lateral proliferation of zoosporangia, formation of zoosporangia next to and below the previous zoosporangium (Figs. 30).There are variations in the amount of time that the two types of zoospores spend in each stage. In Achlya, the primary zoospore is very short lived. As soon as the zoospore is released from the zoosporangium, it immediately encysts. When the cyst germinates, the secondary zoospore is released (Figs. 30-31). Another variation in which the primary zoospore is further suppressed is in Dictyuchus (Fig. 32). The primary zoospores encyst within the zoosporangium and are not released. When the zoospores encyst, they expand and press each other, leaving no intercellular space. As a result, when the secondary zoospores are released from the zoosporangium, a net-like pattern can be seen in the now empty zoosporangium. See the videos below figure 7 to view primary zoospore release in Saprolegnia, and immediate encystment of primary zoospores following release from zoosporangium, in Achlya.


Figure 27: From left to right, primary and secondary zoospores of Saprolegnia.

 

Figure 28: Internal proliferation of zoosporangium of Saprolengia. Figure 29: High magnification of Internal proliferation.
Figure 30: Lateral proliferation of zoosporangium of Achyla. Figure 31: High magnification of lateral proliferation.
Figure 32: Dictyuchus zoospores encyst wiithin zoosporangium. Encysted zoospores press against each other, leaving no intercellular space. When I zoospores released, net-like pattern can be seen in zoosporangium (on left). http://protist.i.hosei.ac.jp/pdb/images/Eumycota/Dictyuchus/Dictyuchus.jpg


Videos of zoospore release of Saprolegnia and Achlya, respectively:

YouTube Video


YouTube Video


Sexual Reproduction: Two sexual organs are borne on the mycelium of Saprolegnia. The female structure is the oogonium (Fig. 33) that contains from 2 to 20 eggs and typically have several male structures, antheridia, attached to the oogonial wall at maturity. Sperms of Oomycota are naked nuclei and are not flagellated. Sperm nuclei are are injected directly to the eggs and form the zygote. The egg and sperm are the products of meiosis and are the only haploid stage in the life cycle.

Figure 33: Oogonia and Eggs of Achlya, with attached Antheridia. Note that there are more than a single egg in each oogonium, a characteristic of the Saprolegniales.


Phytophthora palmivora:

Asexual Reproduction: The Zoosporangium (Figs. 34-36) is typically broad-ellipsoid and well differentiated from the vegetative mycelium in this species. Because the zoosporangia are readily deciduous, they will usually not be attached to the mycelium when mounted on a microscope slide for observation. Only the secondary zoospore is produced in this species. The micrographs below were taken under phase interference optics, which is responsible for the dark appearance of the images. Active release of secondary zoospores from zoosporangia of Phytophthora may be viewed in video below figure 10.

Figure 34: Intact zoosporangium of Phytophthora palmivora before release of zoospore. Note that it is morphologically differentiated from the hyphae, unlike those produced by Achyla and Saprolegnia.
Figure 35: Zoosporangium of Phytophthora palmivora with zoospores clustered together and oozing out of operculum.
Figure 36: Zoospores of Phytophthora palmivora coming apart. Two zoospores swimming away can be seen at the top of the screen.

Secondary zoospore release from zoosporangia of Phytophthora palmivora:

YouTube Video


Sexual Reproduction: The oogonia in this order, unlike those in the Saprolegniales contains only one egg/oogonium (Fig 37).

Figure 37: Oogonium of Peronosporales. Single egg in oogonium. From http://www.fungid.info/Fungid/


Literature Cited

 

Kreisel, H. (1969). Grundzüge eines natürlichen Systems der Pilze. Cramer.

Pringsheim, N. 1858. Beiträge zur Morphologie und Systematik de Algen II. Die Saprolegnieen. Jahrb. Wiss. Bot. 1:284-304.

Tyler, B.M., et al. 2006. Phytophthora genome sequences uncover evolutionary origins and mechanisms of pathogenesis. Science 313:1261–1266.

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