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Consortium for Educational Communication Module on Mucor- I mportant Features and Life Cycle By Dr Masood Majaz Ganaie Asstt. Prof. Degree collage Pulwama Cell No: 9419920786 email:[email protected] Consortium for Educational Communication Text Taxonomic Description Mucor L. (1953), also called as black mould or pinmould, belongs to the subdivision Zygomycotina, class Zygomycetes, order Mucorales and family Mucoraceae (Alexopoulos et al., 1996). The fungi included in Zygomycotina are mostly terrestrial, and reproduce asexually by nonmotile spores. Their thallus is usually mycelial, aseptate and the cell wall contains chitin and chitosan. They are normally haploid. The principal characteristic that distinguishes class Zygomycetes is the production of a thick-walled resting spore called zygospore (Alexopoulos et al., 1996). The zygospore develops within a zygosporangium that is formed after fusion of two gametangia during gametangial copulation. However, because not all the species placed in this class have been shown to produce zygopspores, a variety of morphological, biological and ecological characteristics also enter into defining the Class. These include usual presence of a coenocytic mycelium, asexual reproduction usually by sporangiospores, and absence of flagellate cells and centrioles. The class Zygomycetes is differentiated from the related class Trichomycetes, in having their mycelium not immersed in the host tissue. The order Mucorales has by far the largest number of species and morphological diversity within the class Zygomycetes (Alexopoulos et al., 1996). Mucoralean species have a well-developed mycelium that is generally aseptate. When septa are present, they lack pores with specialized plugs. This character separates them from Dimargaritales and Kickxellales (Alexopoulos et al., 1996). Mucorales can also be separated from other orders of Zygomycetes by their relatively non-specialized associations with other organisms, besides a combination of their asexual and sexual reproductive structures. Most of the genera of Mucorales are saprobes that occur in dung, soil, humus and other organic debris. Typically they have a well-developed mycelial thallus. The mycelium is coenocytic except in family Sigmoideomycetaceae. The thallus exhibits an active cytoplasmic streaming. Anastomoses between the highly branched somatic hyphae Consortium for Educational Communication are rare in Mucorales and as a result, these fungi do not form the interconnected network, typical of most of the fungi (Ingold, 1978). Hyphal septa usually are produced only to delimit sporangia or gametangia or old or injured hyphae. Multiporate, gametangium-delimiting septa are present in a number of species (Benny and Benjamin, 1992) and have been suggested as being typical of mucoralean species (O’Donnell et al., 1997). The order Mucorales reproduces asexually by sporangiospores or rarely by conidia. The outer walls of zygospore are formed by modification of gametangial walls. Mucoraceae is one of the largest families of Mucorales. The genera belonging to Mucoraceae produce non- apophysate sporangia with either deliquescent or persistent walls. There is often a slight constriction of the sporangiophore immediately below the sporangium. Zygospores have opposed, nonappendaged suspensors. Mucor is a large genus with more than 60 species and in general has a cosmopolitan distribution. It can be isolated from almost any organic material that is in contact with air, so can be found in soil, decaying plant material, dung, air (M. racemosus and M. mucedo), and even in certain cases as a parasite on other fungi. However, M. amphibiorum is currently limited to Tasmania. Most of the species are saprobes on a variety of materials including food stuffs. Some are week parasites of fruits and vegetables. The genus Mucor can be differentiated from Absidia, Rhizomucor and Rhizopus (related genera) by the absence of stolons and rhizoids. Structurally Mucor can be differentiated from Rhizopus in having less mycelial differentiation and sporangiophores arising from any point rather than at specific points (as in later one). Unlike Zygorhynchus (Mucoraceae), most of the species of Mucor and Absidia (Mucoraceae) are heterothallic. The molecular phylogeny reveals that the species of Mucor do not form a monophyletic clade and considerable variation can be seen even within species. The majority of species can be placed into one of three groups based on their morphology. These are the M. hiemalis group, which consists of a number of morphologically similar species. The other two groups are the M. circinelloides-group characterized by rather small Consortium for Educational Communication species with reddish-brown zygospores (Schipper, 1976), and the M. mucedo group, characterized by the tall group species, which generally show optimal development at temperature below 20° C (Schipper, 1975). Traditionally, Mucor species were grouped according to morphological similarities, although these groups do not necessarily reflect the phylogenetic relationship among species (Schipper, 1973). This was confirmed by O’Donnell et al. (2001) and White et al. (2006), who found that the monophyletic groups based on the Elongation 1-α gene 28 sequences do not reflect family relationships within the Mucorales. However, data from this gene region can effectively be used to identify undescribed species. The branching of sporangiophores (branched or unbranched), the shape of the sporangiospores (round or elongated), maximum temperature of growth, presence of chlamydospores, assimilation of ethanol, and molecular analysis aid in differentiation of Mucor species from each other. Structure The thallus is eucarpic and mycelial. Colonies of this fungus are typically white to beige. Older colonies become grey to brown in colour due to development of spores. These are very fast growing and on culture medium may grow to several centimeters in height. The hyphae are coarse, coenocytic and richly branched, with branches usually tapering to fine points. Septa may develop at later stages or to separate the older or injured parts. The mycelium growing on substratum can be distinguished into i) absorption hyphae which penetrate the substratum and absorb food, and ii) sporangiophores, the bulk of aerial hyphae. The species produce non-apophysate sporangia that have a wet or dry wall when mature. The suspensors are typically enlarged, equal and opposite. Cell walls are complex with chitin micro-fibrils and abundance of chitosan. Sporangiophore walls are mainly composed of polysaccharides with smaller amounts of lipids and proteins. Four distinct polysaccharides were recognized within the wall: an acid-soluble polymer of D-glucosamine (chitosan), an alkali-soluble hetero-polymer of D- glucuronic acid or fucose or mannose or galactose (mucoran), and two insoluble Consortium for Educational Communication polymers, one of N- acetyl-glucosamine (chitin) and the other of D- glucuronic acid (mucoric acid). Sporangiophore walls resemble the hyphal walls in qualitative composition but differ appreciably in quantitative composition. The cytoplasmic contents contain polysaccharides, proteins, pyrimidines; Mg and Ca have also been detected. It was in M. rouxii that chitosomes were first time reported. The cytoplasm often shows rapid streaming; nuclei are irregular in shape, which divide by constriction and not by mitosis and spindle formation. In anaerobic liquid cultures, especially in presence of CO2, its mycelium breaks down in to small, spherical and independent cells. This is called ‘torula stage’ or ‘torula condition’. When first discovered, it was thought to be a different fungus altogether and was given the name Torula. Then it was found out that it is just an abnormal condition in Mucor. The condition reverts back to filamentous state in presence of O2. Growth Pattern Colonies of Mucor grow rapidly at 25-30°C and quickly cover the surface of the agar in culture medium. Its fluffy appearance, with a height of several centimeters, resembles cotton candy. From the front, the color is white initially and becomes grayish brown with time. From the reverse, it is white. Mucor indicus is an aromatic species and may grow at temperatures as high as 40°C. Mucor racemosus and Mucor ramosissimus, on the other hand, grow poorly or do not grow at all at 37°C or above. Genetic Constitution and Nuclear Behaviour Many of the genes and proteins of Mucor have been sequenced. Sosa et al. (1999) resolved the structural details of a ribosomal protein gene in Mucor racemosus. It consists of two exons of 57 and 387 nucleotides. The protein predicted from the nucleotide sequence contains 148 amino acids and exhibited 61% identity with the S19 ribosomal protein of Xenopus laevis. Two Mucor circinelloides structural genes involved in isoprenoid biosynthesis have been isolated and characterized. The isoA gene encodes a typical eukaryotic farnesyl diphosphate synthase (EC 2.5.1.10), whereas the isoB gene deduced amino acid sequence shows similarity to fungal medium- Consortium for Educational Communication chain prenyl diphosphate synthase. Papp et al. (1999) calculated size of the mitochondrial genome of Mucor piriformis to be about 33.53 kbp. The deduced primary translation product of gene encoding the aspartyl protease of M. miehei contains an N-terminal region of 69 amino acid residues not present in the mature protein. The M. circinelloides gene encoding glyceraldehyde-3-phosphatedehydrogenase (GDP) has also been isolated and analyzed (Acs, 2003). The complete nucleotide sequence encodes a putative polypeptide chain of 339 amino acids interrupted by 3 introns. The predicted amino acid sequence of this gene shows a high degree of sequence similarity to the GPD proteins from other filamentous fungi. The predicted amino acid sequence of this gene shows a high degree of sequence similarity to the glyceraldehyde-3- phosphate dehydrogenase proteins from yeast and filamentous fungi (Papp et al. 2003; Vastag et al. 2003). A 4.4 kb PstI restriction endonuclease fragment of Mucor circinelloides DNA has previously been shown to both complement a leuA− mutation, and to enable the autonomous replication of plasmids within this organism. The complete nucleotide sequence of this fragment has been determined and an open reading frame of 1935 bp with no introns has been identified, which exhibits significant similarity (75% at the nucleotide level) with 114 bp of the 5’ coding region of the Saccharomyces cerevisiae LEU1 gene (Isabel et al., 1989) In Torula stage, all the cells were multinucleate, often containing several dozens of nuclei, while most buds possessed between 2 and 10 nuclei. Up to a number of 35 nuclei, a linear relationship exists between the nuclei number and the cell size (radius). During mitosis in Mucor hiemalis, the chromatin occupies the periphery of resting as well as dividing nuclei. It could not be resolved into separately visible chromosomes and was never seen arranged in the form of a metaphase plate. Division of the nucleus, and of the nucleolus within, is achieved by elongation followed by constriction. The unvarying optical contrast between nucleoplasm and cytoplasm during division implies that Consortium for Educational Communication mitosis takes place within the intact nuclear envelope. Electron microscopy reveals that the division is accompanied by the development of a short, straight, slender bundle of microtubules ( spindle ) in one corner of an elongated nucleus. The spindle extends between largely intranuclear spindle pole bodies . It rapidly increases in length, and in maximally elongated nuclei, runs in a straight line between the two poles, almost completely embedded in nucleolar material. It is tentatively proposed that the chromosomes are attached to the poles of the spindle. The shortness and peripheral, eccentric position of developing spindles in already considerably elongated nuclei suggests that the spindles do not actively initiate nuclear elongation but follows it passively. Its function is seen as that of a device for keeping daughter sets of newly formed chromatids rigidly apart during the course of their distribution to daughter nuclei. Reproduction The fungus reproduces both asexually and sexually. A. Asexual reproduction Following are the methods for asexual reproduction: Fragmentation, chlamydospores and sporangiospores. 1. Fragmentation: Due to mechanical injury, the vegetative hyphae break up into smaller fragments, and each fragment develops into a new mycelium. 2. Chlamydospores: These are formed under unfavourable conditions. In anaerobic conditions, tips of the hyphae become septate. Each segment secretes a thick wall and assumes a round shape. These are called chlamydospores. They can survive in unfavourable conditions, and germinate to form a new mycelium when conditions become favourable. Arthrospore formation in Mucor, the process of septation and fragmentation of hyphae into individual new entities, is not very well understood and often referred to Consortium for Educational Communication as a mere starvation response (Orlowski 1991). Their development is stimulated by specific conditions, including high spore inoculum concentrations and low glucose concentrations. The process of arthrospore formation by M. circinelloides take place more rapidly than in other fungi. 3. Spores (aplanospores or sporangiospores): This is the most common method of asexual reproduction. The sporangia develop terminally on hyphal branches, known as sporangiophores. Sporangiophores are hyaline, erect and simple or branched (e.g. in M. proliferus). In M. renisporus, tall sporangiophores are mostly un- branched, while short sporangiophores are sympodially branched. They bear large (60-300 µm in diameter), terminal, multispored sporangia, which are without apophysis, but have welldeveloped subtending columellae. In case of the species with branched sporangiophores, terminal sporangium is larger than the lateral ones. In M. renisporus, collumellae are cylindrical to pyriform in shape. The smaller columellae are mostly conical. Yellow lipid droplets are present in most columellae as has been reported in M. renisporus. A conspicuous collarette (remnants of the sporangial wall) is usually visible at the base of the columella after sporangiospore dispersal. However, the collarettes are absent on short sporangiophores in M. renisporus. The sporangiophores of M. grandis only posses a short row of spines in place of a collarette. Sporangiospores are hyaline, grey or brownish or dark, globose to ellipsoidal, and smooth-walled or finely ornamented. However, in Mucor renisporus they may be kidney-shaped in side view. In Absidia, a related genus, the sporangia are pear-shaped. There is considerable inter- and intra- species variation in spore size (Taeko, 1974). Large spores are clearly multi-nucleate. During the process of sporogenesis, apex of the sporangiophore swells and cytoplasmic mass along with nuclei move in this part. The swollen part enlarges into a large globose structure. This is the young sporangium. On maturity, contents of sporangium become differentiated into a thick, dense layer of cytoplasm, with many nuclei Consortium for Educational Communication towards the distal region beneath the sporangial wall, and a vacuolated subglobose and nuclei free portion towards the centre. An extension of that sporangiophore, called columella, protrudes into the sporangium. A dome- shaped septum is then laid down, cutting off a distal, peripheral portion (which will contain the spores) from a central cylindrical or subglobose spore-free core, the columella. The contents of the distal portion become cleaved into multinucleate spores. However, spores are uninucleate in certain species such as M. hiemalis. All the spores are almost of the same size but in M. proliferus, the spores formed in the terminal sporangia are larger than those produced in the lateral sporangia. When the sporangia are mature, the sporangiophores may be seen as coarser, blunt-tipped aerial hyphae growing away from the substratum. The sporangial wall dissolves, except for basal region, where its remnants can be seen as frill or collar at the base of the columella. The spores remain adhered to columella and are not easily disseminated, usually mites bring about dissemination. In contrast to Mucor, in Rhizopus spores are easily blown away by wind. Columella after dehiscence of sporangium does not change shape in Mucor. While columella in most of the species of Rhizopus become more or less dome- or umbrella- shaped after dehiscence of sporangium. In M. plembelis, the sporangial wall breaks into pieces. The wall of mature sporangiospore consists of an inner, electrontransparent layer and an outer electron dense layer. The spore wall is covered with two thin layers, each about 10 nm thick, which may correspond to ordinary spore sheath. The cell membrane of the spores do not have invaginations like those of higher fungi. Instead, there are numerous round depressions about 50 nm in diameter. Mitochondria of the spores are much larger and show wide and deep invaginations of their membranes. Their cristae are indistinct. Lipid droplets have multi-layered shells and are much more highly developed than those found in mycelial cells, Torula stage or chlamydospores of this fungus. When they fall on suitable substratum, in presence of proper moisture and temperature, they germinate by producing g nerm- tubes, which then establish new mycelia. Sexual reproduction Consortium for Educational Communication Sexual reproduction in Mucor is of isogamous type, i.e. the two mating gametes are morphologically identical. Some species are homothallic (e.g. M. genevensis), but most of them are heterothallic (e.g. M. hiemalis). The conjugation of two gametangia gives rise to a zygospore. In heterothallic species, zygospores are formed only when gametangia of compatible strains contact each other. If the appropriate strains are inoculated at opposite sides of a Petri dish, the gametangia grow out and a line of zygospores develops in the centre where these mycelia meet each other. In the heterothallic species of Mucor, as in M. mucedo and M. hiemalis, the sexual act is initiated when hyphal branches of (+) and (-) strains contact each other. Initiation of gametangia is believed to be induced by the trisporic acids. They are produced in appreciable amounts only when (+) and (-) cultures are in continuous diffusion contact with each other. Actually the strains first produce small quantities of pheromones that are converted to trisporic acids. However, Mesland et al. (1974) reported that the vegetative mycelium of M. mucedo, even when not in diffusion contact with vegetative mycelium of opposite strain, could produce volatile substances which induce attraction of the compatible gametangia (zygotropic response). These volatile substances have a dual role- to enhance trisporic acid synthesis in the opposite mating type, and to mediate the zygotropic response. In homothallic species, zygophores of the same thallus can fuse to form a zygosporangium. Each branch (zygophore) swells at the tip to develop a progametangium. Dense cytoplasm and numerous nuclei flow to the contacting tips which enlarge further. A septum then separates the terminal part called gametangium from the remaining part of the progametangium, the suspensor. When the compatible and mature gametangia contact one another, their tips swell, and fuse apically to form a fusion septum. The fusion septum (separating wall) dissolves and the protoplasts of both mix with each other. The structure formed by the fusion of two gametangia, enlarges, develops a thick wall, and is called zygosporangium. The plasmogamy is followed by nuclear fusion (karyogamy) of the two compatible strains to give numerous diploid nuclei. Secondary wall Consortium for Educational Communication material is deposited, causing the zygosporangium wall to thicken and become pigmented. Localized thickening results in the formation of the ornamentation characteristic of each species. The mature zygosporangium is more or less globose. The plasmogamy and karyogamy within the zygosporangium gives rise to a resting spore, called zygospore. However, in certain conditions zygospores may also be formed parthenogenetically, and is called azygospore as has been reported in M. bainieri (Ginman and Young, 1993). The zygospore is hyaline and characteristically with a single, eccentric globule and many diploid nuclei. Meanwhile the zygospore enlarges and secretes a thick wall, which is differentiated into exospore and endospore. The zygospores are usually brown to black; with rough blunt projections or warts. The wall of mature zygospore is probably five-layered. The endospore is made up of three thin inner elastic layers, whereas the exospore consists of two brown or black outer fragile layers. As the zygospore matures, the wall of the zygosporangium breaks up into fragments, which fall apart. All the diploid nuclei, formed in the zygospore undergo meiosis usually before the germination. The zygospore germinates usually after a long period of rest. During germination, the outer wall cracks and the inner wall comes out in the form of germsporangiophore. All the nuclei in the zygospore migrate to the tip of the germ-sporangiophore. Consequently, it swells to form a sporangium called germ- zygosporangium. Rarely more than one germ-sporangiophores arise from a single zygospore. However, occasionally the germsporangiophores may be branched and in such cases more than one sporangia are present on each germsporangiophore. Each of which bears a single terminal germ-sporangium containing many spores. Life Cycle In asexual phase, Mucor reproduces either by means of vegetative propagation or by means of spores. Many a times due to mechanical injury, the vegetative hyphae may break into smaller fragments, and each fragment regenerates to form a new mycelium. Additionally, under unfavourable conditions, the tips of the hyphae become Consortium for Educational Communication septate. Each septate region secretes a thick wall around itself to form a chlamydospore. On the arrival of favourable conditions each such spore germinates to form a new mycelium. However, the most common method of asexual reproduction is by sporangiospores. They develop within sporangia, arising on the tips of sporangiophores. Contents of the mature sporangium become differentiated into dark, elliptical or ovoid spores. The dehisced spores, after falling on suitable substratum, in presence of proper moisture and temperature, germinate to form new mycelia. In the asexual phase, all the stages are haploid. In sexual phase, Mucor reproduces by means of isogamy and the most of species are heterothallic. The compatible gametophores contact with each other and their distal segments fuse to form zygosporangium. The plasmogamy is followed by karyogamy and a brief transitory diploid stage is established. The contents of the zygosporangium develop into a spore called zygospore. The zygospore germinates after a long period of rest. Before the germination diploid nuclei formed in the zygospore undergo meiosis. Thus haploid stage is reverted back. All the nuclei in the zygospore migrate to the tip of the germinated zygosporangium. The contents of the zygosporangium differentiate into spores, which in turn, germinate to establish a new haploid mycelium. Thus the life cycle of Mucor is haplontic. Dimorphic Mucor species are capable of growth as either aseptate filamentous mycelial form, or as yeastlike form. The gaseous atmosphere constitutes a pivotal factor in determining the type of the form. The hyphal form predominates when conditions are aerobic, whereas strict anaerobic condition is required for the development and maintenance of the yeast- like form. Furthermore, the nature of the carbon source is crucial: while the yeast like form can only grow on fermentable hexoses, hyphal form has been described on a wide range of substrates including complex carbon sources. The exact requirements of each Mucor species to grow in a particular mode may vary. Mucor circinelloides is able to grow in a filamentous mode on a wealth of carbon sources. However, not only Consortium for Educational Communication anaerobic condition but also a concentration of 30% CO2 in the sparging gas are necessary to facilitate its yeast-like growth. Experiments have shown that new buds emerge at random locations all over the cell surface. During germination more than one germ tubes may emerge from a spore. This is more likely to be the case with xylose than with glucose as a carbon source. The formation of new branches occurs randomly. Comparatively larger number of tips per hyphal length is formed in presence of xylose than on glucose. M. piriformis is a soil-borne fungus that is found mainly in the surface layers of orchard soil. It infects fallen fruit during and after harvest, and as a result orchard populations are highest in the months immediately following harvest. In the orchard, the fungus may be spread by rain and irrigation splash, by bird and insect feeding, and by practices such as mowing which can spread infected pieces of fruit. Economic Importance The genus Mucor is of considerable economic importance (Domsch et al. 1980). Many species of Mucor are used in industrial fermentation at the initial stages, for converting starch into sugar. Mucor circinelloides is proving to be a promising cost-effective biofuel (Vicente et al., 2010). Amylase, cellulase and oxido- reductase enzymes are being obtained from Mucor. Mucor circinelloides is a multifaceted organism, capable of growing on a diverse range of substrates, possessing the ability of multiple modes of growth and having the potential of industrial application for the production of both degradative enzymes and primary metabolites. The growth capabilities of the organism facilitate its attractiveness as an industrial workhorse. Mucor circinelloides produces an extracellular enzyme polygalacturonase, which can be of tremendous economic importance (Thakur et al., 2010). Certain species have been used for lupeol transformation (Carvalho et al., 2010) and production of enzymes like glucose isornerase. Mucor pusillus is being used for production of chymosin (Wei-Dong et al., 2010). M. racemosus has been used for the production of rennin. Consortium for Educational Communication Most of the species are unable to infect humans. Most infections reported list M. circinelloides and similar species such as M. indicus (M. rouxii), M. ramosissimus and M. amphibiorum as the causative agents. However, M. hiemalis and M. racemosus have also been reported as infectious agents, although their inability to grow at temperatures above 32 ₒC raises doubt as to their validity as human pathogens and their pathogenic role may be limited to cutaneous infections (de Hoog et al. 2000). Some thermo-tolerant species sometimes induce rapidly spreading necrotizing infections, known as zygomycosis. Deja et al. (2006) found M. indicus involved in gastro-intestinal zygomycosis. While M. circinelloides was identified as causative agent of primary cutaneous zygomycosis (Chandra and Woodgyer, 2002 and Irwin et al., 2008) and invasive maxilla-facial zygomycosis (Khan et al., 2009). Similarly epidermal necrosis was found to be complicated by Mucor infection by Jin et al. (2008). Some species of Mucor attack stored grains. M. amphibiorum is the only pathogen known to cause significant morbidity and mortality in free living platypus in Tasmania (Connolly et al., 2010). It causes ulcers in it, which can be secondarily infected and potentially fatal. It can also reduce the ability to regulate body temperature. Mucor species are mostly known as spoilage organisms, and only one species (M. piriformis) has been reported as a pathogen on stone- and pome- fruit (Michailides and Spotts, 1988). Mucor rot is a post- harvest disease of apples, pears and other fruits caused by the fungus Mucor piriformis and less often by other Mucor species. The disease is found in Australia, North America, South Africa and Europe, where it can cause serious losses, especially after long- term storage of fruit. Mucor rot causes a light brown, soft, watery rot. It commonly occurs where the fruit skin has been damaged and also at the stem and calyx end of fruit. Where breaks in the fruit skin occur, white, whiskery fungal growth may appear which is soon covered by black spore masses. The rot progresses rapidly at room temperature and even at storage temperature (0°C). The disease Consortium for Educational Communication can cause extensive rotting over several months. Disease symptoms are superficially similar to those of rhizopus rot caused by Rhizopus stolonifer; however, mucor rot can develop at 0°C whereas rhizopus rot is restricted by temperatures below 4°C. In Australia, mucor rot has only relatively recently been recognized as a serious problem. In some seasons considerable losses have occurred, especially in CA-stored fruit for both local and export markets. Spread within a packing shed can be rapid, as healthy fruit can be contaminated and infected during post- harvest dipping, and during sorting and grading operations. M. piriformis can also be a major cause of core rot in Red Delicious apples. M. piriformis enters the packing shed on soil adhering to fruit bins and machinery, and on fruit that was picked off the ground during harvest. It may also survive between seasons on fruit storage bins. Inoculum can be spread during post- harvest handling operations, for example, through drench and flotation solutions. During storage, rotted fruit break down and release juice which, in the case of pears, assists in secondary spread. Secondary spread during storage is less common in apples.