Anaerobic Fungi

Biology: Ecology, and Function

Douglas Mountfort, Mountfort

• Publisher: CRC Press
• Year: 2020
• Topic: Biological Sciences
• eBook ISBN:
  9781000148176
• Subtopic: Biology
• Index: Biological Sciences

Uniting-for the first time-current information on anaerobic fungi from a number of different disciplines, this unique reference examines the taxonomy, physiology, biochemistry, molecular biology, and ecology of anaerobic fungi-focusing on fungi from the rumen and other gut environments such as the cecum and hindgut of nonruminant herbivores.
Anaerobic Fungi

Presents new techniques for culturing anaerobic fungi!

analyzes the isolation, culture, and survival of anaerobic fungi

describes the nucleic acids of anaerobic fungi, gene cloning, and the establishment of molecular phylogeny

discusses the fermentation of carbohydrates

explains how anaerobic fungi interact with other microorganisms

investigates the ultrastructure of plant cell walls degraded by fungi

details the effects of diet on fungal populations

delineates specific procedures for quantifying anaerobic fungi

outlines potential directions for future research in molecular genetics

and more!

1

Anaerobic Fungi: Taxonomy, Biology, and Distribution in Nature

COLIN G. ORPIN*

Division of Tropical Crops and Pastures
CSIRO
Brisbane, Queensland, Australia

I. THE DISCOVERY OF ANAEROBIC FUNGI

Herbivorous mammals depend for their survival on a symbiotic association with microorganisms in their alimentary tract. The diet of grazing and browsing herbivores consists largely of plant structural carbohydrates, such as cellulose and hemicelluloses, that the animals themselves are unable to digest. Instead, the symbiotic microorganisms in the alimentary tract, particularly in the rumen of ruminants and the cecum and hindgut of nonruminants, hydrolyze these compounds under anaerobic conditions, with the production of microbial cells and volatile fatty acids that the animal can utilize as sources of nutrition (1,2,3). In order to understand and control digestion of plant structural carbohydrates and improve the production efficiency of ruminants, the microbial population of the rumen of domestic sheep and cattle has been studied extensively (1,2,3).

The microbial population of the rumen is diverse, and until the discovery of anaerobic fungi, it was believed to consist principally of anaerobic and facultatively anaerobic bacteria, ciliated protozoa, and flagellate protozoa. Early workers (4,5) documented the existence of uniflagellate, biflagellate, and multiflagellate organisms in rumen contents, believing them to be flagellate protozoa. These organisms were placed in the genera Callimastix, Oikomonas, Monas, and Sphaeromonas. Multiflagellate organisms similar to the Callimastix frontalis described by Braune (5) in the rumen were subsequently found in different habitats; Callimastix equi Hsuing was found in the horse cecum (6), Callimastix jolepsi in the pulmonate snail (7), and Callimastix cyclopis in the copepod Cyclops stenuus (8). The status of the multiflagellate organisms from the horse cecum and the pulmonate snail remains to be determined, but Callimastix cyclopis has been examined in some detail (8). It was found that the flagellate was in fact a zoospore of a fungus with a plasmodial vegetative stage that developed in the body cavity of the host copepod and, on maturity, gave rise to the flagellates. It was believed that the flagellates would then infect a new host to continue the life cycle. Since this species was identified as a fungus and not a protozoan, it was proposed that the rumen flagellates with multiple flagella, which were still assumed to be flagellate protozoa, should be reclassified as a species of zooflagellate in the new genus Neocallimastix (8), with frontalis (5) as the type species.

Many of the flagellated rumen organisms described by Liebetanz (4) and Braune (5) have since been shown to be not protozoa, but the flagellated zoospores of anaerobic fungi (Fig. 1). The first report of isolation of an anaerobic fungus, a species of Neocallimastix, was published in 1975 (9). The organism was isolated during attempts to isolate and culture anaerobic flagellate protozoa from the rumen contents of sheep using a published procedure (10). Flagellates did indeed grow in the cultures, but it was impossible to separate the flagellates from what appeared to be vegetative fungal growth in the culture. It was soon evident that the flagellates were released from reproductive structures borne on the fungal rhizoids, and that the life cycle of the organism consisted of an alternation between a motile, flagellated zoospore stage and a vegetative rhizoid-bearing reproductive stage. The organism was similar both in morphology and life cycle to a chytridiomycete fungus, but it was a strict anaerobe. Until then, fungi were regarded as being either anaerobes or facultative anaerobes, and the detection of microorganisms similar to chytridiomycete fungi that could grow only under chemically reducing conditions in the absence of molecular oxygen was novel. Because of the revolutionary nature of this finding, acceptance by the scientific community was slow. The presence of chitin in the cell walls of these and similar organisms (11) confirmed that they were true fungi despite being strict anaerobes.

Methods for the isolation and culture of anaerobic fungi have since been considerably refined, so that the organisms can now be routinely isolated from suitable habitats with little difficulty. Anaerobic fungi are now regarded as a normal component of the microbial population of the rumen.

The reason why the rumen fungi remained undiscovered while research on rumen bacteria and protozoa went ahead strongly during the period up to 1975 is not difficult to understand, even though electron microscopy was commonly employed to determine microbial activity during the digestion of plant tissues in the rumen and the flagellates of the rumen fungi had been described (as flagellate protozoa) as early as 1910 (4). Work on the flagellate protozoa of the rumen was limited because of their small population densities and the consequent assumption that they were of little importance in rumen metabolism; hence the fungal flagellates were also largely ignored. In addition, it was common for rumen microbiologists to strain rumen contents through cheesecloth to remove the large plant fragments before microbiological analysis, as pointed out by Bauchop (12). In doing this, the microbiologists were separating the bulk of the vegetative growth of the fungi from their working material, and it was only after the introductory work of Orpin (13,14) on the invasion of plant tissues by the flagellates of the rumen fungi, and the scanning electron microscopy of Bauchop (15), that the significance of separating the plant particles from rumen contents was recognized in this context. The fungal growth is normally tightly attached to digesta fragments (Fig. 2). However, rough handling during the straining of digesta through muslin sometimes damages the vegetative fungal growth and breaks sporangia from the rhizoids (Fig. 2b). These sporangia can be differentiated from protozoa (Fig. 1c) in the filtered rumen fluid by their lack of motility, high refractivity, and lack of cilia; they contain no skeletal plates and usually have a short length of rhizoid attached.

Figure 1 Flagellate zoospores of anaerobic fungi as observed in fresh rumen contents, (a) A multiflagellate zoospore, possibly of Neocallimastix sp., is shown, together with the large bacterium Magnoovum eadii (arrowed), (b) A uniflagellate zoospore is shown, together with Quin’s Oval (Q) and another large bacterium – Selenomonas ruminantium (S). The zoospores can be clearly differentiated from the large bacteria, (c) Uni flagellated zoospores (arrowed) can be seen in relation to a ciliated protozoan, Entodinium sp. (P). Phase-contrast microscopy. (a) × 2000; (b) ×2000; (c) ×700.

Additional reasons why the rumen fungi remained undiscovered until recent years were the difficulty of isolating them from rumen contents without the use of antibiotics to suppress the growth of bacteria (9,14,16,17) and the need to isolate them from low dilutions of rumen contents. This is usually within the range of 10⁻³–10⁻⁵, well below that normally employed for the isolation of anaerobic rumen bacteria. Thus colonies of anaerobic rumen fungi would probably not have been observed during the isolation of rumen bacteria.

The importance of these fungi in herbivore nutrition is still not well understood, but their undoubted ability to utilize major plant cell wall polysaccharides for growth (18–20) and to produce a wide range of enzymes with activities capable of hydrolyzing many of the components of plant cell-walls (21,22,23) shows that they have the potential to contribute substantially to plant-fiber degradation in the alimentary tract of the host animal.

The life cycles of all anaerobic fungi so far described consist of an alternation between a motile, flagellate zoospore stage, free-living in the liquid phase of the digesta, and a nonmotile, vegetative, reproductive stage, saprophytic on digesta fragments in the alimentary tract of the animal. The zoospores of all species are able to change to an ameboid form and may be observed crawling over digesta fragments and, in culture in vitro, the sides of culture vessels. Evidence is accumulating that in some species (isolated from ruminants) an additional oxygen-tolerant stage may be developed in the feces. An oxygen-resistant stage, possibly a sporangium, is suspected (24), but in an isolate from the horse cecum, a possible resting stage unlike a sporangium has been identified (25). In the coelomic parasite Coelomyces ( = Callimastix) cyclopis of Cyclops stenuus, a resting cyst gives rise to biflagellate zoospores that are the infective stage for the mosquito, a second host of the parasite (26); as yet no such cysts have been observed in the anaerobic fungi, unless the putative resting stage of an isolate from the horse cecum (25) is equivalent. A schematic, generalized life cycle of anaerobic fungi from the rumen as we understand it at present is shown in Fig. 3.

Figure 2 Vegetative stages of anaerobic fungi found in rumen contents. In the scanning electron microscope (a), the sporangia of the fungi can be seen with the associated rhizoid (arrowed) penetrating the plant...

Table of contents

• Cover
• Halftitle Page
• Title Page
• Copyright Page
• Series Introduction
• Preface
• Contents
• Contributors
• 1. Anaerobic Fungi: Taxonomy, Biology, and Distribution in Nature
• 2. The Ultrastructure of Anaerobic Fungi
• 3. Nutrition and Survival of Anaerobic Fungi
• 4. Fermentation Product Generation in Rumen Chytridiomycetes
• 5. Regulatory Constraints in the Degradation and Fermentation of Carbohydrate by Anaerobic Fungi
• 6. Ultrastructure of Plant Cell-Walls Degraded by Anaerobic Fungi
• 7. Interactions Between the Rumen Chytrid Fungi and Other Microorganisms
• 8. Effects of Diet on the Fungal Population of the Digestive Tract of Ruminants
• 9. The Nucleic Acids of Anaerobic Fungi
• 10. Differential and Integral Equations and Their Application in Quantifying the Fungal Population in the Rumen
• 11. Anaerobic Fungi: Future Perspectives
• Index