In this article we will discuss about the life cycle of penicillium with the help of suitable diagrams.
Mycelium of Penicillium:
The mycelium is well developed and copiously branched. It is composed of colourless, slender, tubular, branched and septate hyphae. The hyphae run in all directions on the substratum and become intertwined with one another to form a loose network of hyphae constituting the mycelium.
Some of the hyphae may even grow into the interior of the substratum and the rest spread on the surface. The former secrete enzymes and absorb food materials from the substratum. These are the haustorlal hyphae.
The aerial hyphae receive nourishment through the haustorial hyphae and produce reproductive structures. Baker (1944) observed anastomosing between hyphae of two mycelia resulting in a heterokaryotic mycelium.
The mycelium in a few species may develop into a sclerotium. The hyphae constituting the mycelium are septate and the cells are short. The septa between the cells have each a central pore. Through the pores the protoplasm flows from cell to cell.
Reproduction in Penicillium:
Penicillium reproduces both asexually and sexually. The asexual stage however, is dominant and constitutes the usual mode of reproduction. Sexual stage is rare.
1. Asexual Reproduction:
It takes place by vegetative methods and sporulation.
(i) Vegetative Reproduction:
It is accomplished by the most common method of fragmentation. The hyphae break up into short segments. Each segment or fragment grows by repeated division into a full-fledged mycelium.
In some species, the mycelium forms compact resting bodies, the sclerotia. Sclerotia enable the species to survive periods of stress or to hibernate. On the onset of conditions favourable for growth each sclerotium germinates to form a new mycelium. The sclerotia thus serve primarily as a means of perennation rather than multiplication.
Normally it takes place by the formation of non-motile, asexual spores, the conidia which are produced exogenously at the tips of long, erect special septate hyphae called the conidiophores. Penicillium multiplies repeatedly by this method during the growing season.
Conidiophores (Fig. 10.10 A-C):
A conidiophore arises as an erect, tubular hyphal outgrowth from any cell of the mycelium and not from a specialised cell (foot cell) as in Aspergillus. After some period of vegetative growth upright hyphae arise from the older portions of the mycelium.
They are negatively geotropic and arise singly from any cell of the mycelium. Each grows up in length vertically. Reaching a certain height the septate conidiophore branches once or twice or even more times.
These are termed as primary, secondary or tertiary branches, respectively. Only rarely are the conidiophores unbranched (Penicillium thomii). The unbranched axis of the latter bears a tuft of flask-shaped sterigmata (A).
In the species with branched conidiophores, the ultimate branches which bear tufts of flask-shaped sterigmata or the phialides are called the metulae. The lower branches which support the metulae when short and form a part of the penicillus are called the rami.
Conidia are abstricted from the tips of the phialides or sterigmata and are borne in long, unbranched chains. Baker (1944) reported that the phialides and the upper cells of the conidiophore are uninucleate.
The phialide wall is electron-transparent with a thin electron opaque surface layer. The apical portion of the conidiophore with its branches (metulae), sterigmata and chains of conidia looks like a small artist’s brush known as the penicillus.
The generic name Penicillium is derived from ‘penicillus,’ which reflects the form of conidial chains arising from the sterigmata supported on the metulae.
Development of Conidia:
The conidia are formed within the narrow tips of the flask-shaped phialides (A). The conidium initial is formed by the distension of the tubular tip of the phialide (B). The phialide nucleus undergoes mitosis. One daughter nucleus remains in the phialide and the other migrates into the swollen tip (conidium initial). The conidium initial protoplast is then cut off from the phialide protoplast by a thin perforate septum (C).
The perforation remains as a channel between the successive conidia in the chain. It is filled with electron-opaque material. The newly delimited conidial protoplast secretes a wall around it distinct from the phialide wall and functions as the first conidium (D).
The spore or conidial wall during further development may remain distinct or fuse partially with phialide wall. The tip of phialide below the first conidium again elongates and swells (D). A second conidium chains formed by repeating the process (E).
Like this, one below the other, a long chain of conidia is formed. In older conidium chains, the septum region remains only as a narrow, connecting strand between conidia. The conidium chains are enclosed within an electron-opaque surface layer which appears to be continuous with the surface layer of the phialide wall.
The conidium in the chain are arranged in a basigenous manner. The youngest conidium lies next to the tip of the sterigma and the oldest away from it.
The basigenous arrangement of conidia serves two useful purposes. It permits ready dispersal of mature conidia from the tips. Secondly, it aids the proper nourishment of younger conidia which are nearest to the tips of the sterigmata.
As the conidial chain increases in length, the connectives between the older conidia break down resulting in the separation of mature conidia. The conidia are thus shed continuously. Being small, light and dry they are dispered by air currents.
The conidia under the microscope look like small beads. They may be ovoid globose, elliptical or pyriform. In some species they are smooth and in others rough. Generally the conidia are uninucleate but may become multinucleate in some species.
The conidia may be greenish or pale in colour depending upon the species. It is the spore wall that is coloured. The conidia are responsible for the colony colour characteristic of the species. They serve for quick propagation of the species during the growing season. They are easily disseminated by wind.
Structure of Conidia:
The conidia are tiny spore-like structures globose to ovoid in form. The pigmented spore wall is differentiated into two layers, outer exine and inner intine. The exine is comparatively thick, smooth or spiny. The inline is thin. Under electron microscope it appears to consist of 3 or 4 layers. There is the outermost layer (W1) with an irregular, undulating contour. It is electron-dense.
Empty speaces are often seen between it and the next wall layer. Next to it is a slightly thin inner electron-opaque layer W2 with a more regular contour. This layer (W2) gradually merges into the next wall layer W3 which is thick and electro- transparent. It usually abuts on the plasma membrane. However, in some cases, there is another slightly electron-dense layer (W3) present directly above the plasma membrane. Within the spore wall is the plasma membrane.
Embedded in the cytoplasm of the conidium are the mitochondria and ribosomes. The endoplasmic reticulum strands are not discernible. The vacuoles are absent. However, Martin et al. (1973) reported the presence of a single large vacuole in the resting conidium of P. notatum. The conidium cytoplasm contains oil globules. Usually the conidium contains a single nucleus. The nuclear membrane is two-layered and is poriferous.
The conidial stage in Penicilliutn is more dominant than in Aspergillus. Majority of the species of Penicillium are known only in the conidial stage. Some (about 20 species) are now known to produce cleistothecia.
At first when the connection between the two stages was not fully established the conidial or imperfect stage was given the name form or genus Penicillium and the sexual or perfect stage as Talaromyces. The discovery of sexual or perfect stage in these form- species places them in the true Ascomycete genus Talaromyces.
Many mycologists, however, still adhere to the old name Penicillium because the conidial stage is predominant. Moreover, the generic name Penicillium was introduced first.
Germination of Conidia (Fig. 10.13):
On falling on a suitable substratum and under suitable conditions (moisture, suitable temperature and food), the conidium absorbs moisture and swells. The swollen conidium germinates by putting out a germ tube. Fletcher (1971) studied the fine structural changes during germination of conidia in Penicillium griseofulvum. He reported that the ungerminated conidium has a two-layered spore wall.
The protoplast contains a single nucleus and mitochondria. During germination a third layer appears inside the two-layered spore wall. It is continuous with the germ tube wall. In P. frequentans, the germ tube wall is considered to be an extension of the inner layer of the original spore wall.
During swelling the mitochondria increase in size and become lobed, endoplasmic reticulum becomes visible md vacuoles are formed. The septa formed in the germ tube are perforate and have Woronin bodies associated with them. Marchant (1968) suggested enzymic breakdown of the two-layered spore wall at the point of germ tube emergence rather than mechanical rupture.
Martin et al. (1973) reported that during swelling of the conidium (A), the outer layer (W1) of the wall of the conidium becomes broken at several places and separated from the underlying spore wall layer W2 . The smooth surface layer W2 with a regular contour thus becomes exposed. At this stage the swollen conidium pats out a germ tube (B).
During spore swelling the large mitochondria of the resting spore divide to produce smaller mitochondria. The size of the single vacuole decreases. Endoplasmic reticulum reappears in the swollen and germinating spores. The wall of the emerging germ tube has been reported to be continuous with the electron transparent wall layer W3.
The electron-dense wall layer W2 remains at the base of the germ tube and around the spore. The single nucleus of the resting spore divides early in germination. One of these migrates into the germ tube.
With the elongation of the germ tube most of the mitochondria migrate into it and become concentrated near the tip. Small vesicles reappear along the plasma membrane. Some were seen at the tip of the germ tube. Later, a septum is formed at the point of emergence of the germ tube. It is continuous with the transparent layer W3 of the spore wall (C).
During further development, layers W2 and W1 of the hyphal wall are formed by deposition of electron-dense material on the outer surface of the germ tube. The latter elongates and divides by septa. Septum formation is preceded by nuclear division. Each septum has a central pore.
There are Woronin bodies associated with the septa. By further growth, septation and branching mycelium is formed. It consists of uninucleated cells.
The mycelial hyphae of Penicillium, when made to grow immersed in a sugary solution, they divide by additional septa into short uninucleate segments. The latter become rounded and separates as thin-walled spore-like structures called the oidia or oidiospores.
Often the oidia increase in numbers by budding. This is known as the torula stage. The fungus m the torula condition brings about fermentation of sugar into alcohol. This process is called alcoholic fermentation. On a solid medium each oidium germinates to produce a normal mycelium.
Similarly if conidia of Penicillium happen to fall in a sugary solution they fail to produce normal mycelia. Instead each conidium germinates to produce a thin-walled yeast-like cell which starts budding like the oidiospore to produce the torula stage.
2. Sexual Reproduction (Fig. 10.14):
Sexual process in Penicillium has been studied in a few species such as P.vermiculatum (=Talaromyces vermiculatus), P. glaucum, P. brefeldianum and a few others. All of them are reported to be homothailic. Derx (1925) reported heterothallism in one species (P. luteum) but his observation has not been confirmed by subsequent investigators.
The structure of sex organs varies from species to species. In Talaromyces vermiculatus (Penicillium vermiculatum) sexual reproduction is oogamous. It was studied in detail by Dangeard in 1907. The male and female organs are known as antheridia and ascogonia respectively.
The mature ascogonium is a long, erect multinucleate, unseptate, tubular structure. At its upper end it may be curved like the handle of an umbrella (E). It arises as a lateral outgrowth from any cell of the vegetative mycelium. When young the ascogonium is uninucleate (A). As it elongates the single nucleus within it divides and redivides to give rise to a definite number of daughter nuclei which is either 32 or 64 (B).
Meanwhile a slender uninucleate hyphal branch originates either from an adjacent cell of the same hypha which gives rise to the ascogonium or from a separate neighbouring hypha. It is the antheridial branch (C). It grows up coiling loosely around the ascogonium making several turns about it (D).
The distal end of the male branch becomes slightly inflated and is eventually cut off as an antheridium by a septum (E). The antheridium is a short, terminal, club-shaped, uninucleate structure.
It is the union of two protoplasts which brings the compatible nuclei close together in the same cell. The tip of the antheridium comes in contact with the ascogonium. At the point of contact, the double wall dissolves. Through the common pore the two protoplasts come in contact. According to Dangeard, the migration of the male nucleus into the ascogonium, does not take place.
This fact has been confirmed by a number of other workers. Mere contact of the antheridium protoplast stimulates the ascogonium. Beyond that the antheridium plays no role. The female nuclei in the ascogonium, however, arrange themselves in pairs.
The pairing of the female nuclei in the ascogonium is called autogamy. Each pair is called a dikaryon. With the establishment of dikaryons the haplophase ends and the dikaryophase starts in the life cycle.
In some other species of Penicillium, both antheridium and ascogonia are claimed to be functional. The intervening walls between the two sex organs dissolve and their protoplasts come in contact. The male nucleus migrates into the ascogonium. Further details of the male nucleus are not known fully.
The suggestion, however, is that it divides a number of times. The male nuclei then come to lie by the side of the female nuclei, each to each. Each pair of nuclei consisting of one male and the other female is called a dikaryon.
Sexual reproduction has been worked out in P. glaucum. Two short lateral hyphae arise from the mycelium close together and become coiled about each other. One of them is the antheridium and the other ascogonium. Plasmogamy thus takes place by gametangial contact. Dikaryons are established in the ascogonium. This is followed as usual by the septation of the ascogonium into binucleate segments.
Post Plasmogamy or Autogamy changes (Fig. 10.14 E-G):
(i) Septation of Ascogonium:
Plasmogamy or Autogamy is followed by septation of the ascogonium. Each segment has a pair of nuclei. Meanwhile entangled sterile hyphae grow up around the sexual apparatus and form an investment of loosely interwoven hyphae and sexual apparatus and afford protection to the structures developing within (E-F).
(ii) Development of Ascogenous Hyphae (Fig. 10.14 F):
As a result of the stimulus of plasmogamy or autogamy one or more lateral outgrowths arise from some of the binucleate segments situated in the middle of the septate ascogonium. Each outgrowth is called an ascogenous initial.
It is binucleate. Each ascogenous initial develops into a branched ascogenous hypha composed of binucleate cells. The branches are of different lengths. In some other species of Penicillium the lateral branches of ascogenous hyphae are one-celled.
(iii) Formation of Asci:
According to Emmons (1935), asci in many species are developed directly from the binucleate cells toward the tips of the branches of the ascogenous hyphae. Consequently they are arranged in short chains. In P. vermiculatum almost all the cells of the ascogenous hyphae develop into asci directly without crozier formation. Rarely the asci are formed from the croziers.
1. Direct Method of Ascus Development:
This is illustrated by P. vermiculatum (Fig. 10.15). All the binucleate cells of the ascogenous hyphae are capable of directly developing into ascus mother cells. No croziers are formed. The binucleate cell enlarges into a sac-like structure. It is the ascus mother cell.
It is globose or pear-shaped in form. The two nuclei of the ascus mother cell eventually fuse. The cell containing the fusion nucleus (synkaryon) is called the young ascus.
2. Indirect (crozier) method of ascus formation (Fig. 10.16):
In this case, the terminal binucleate cells of the ascogenous hyphae or their branches curl over to form a hook-like structure called the crozier. For further details of the process refer to prior pages.
The binucleate cell in both cases enlarges to function as the ascus mother cell. It is the last structure of the dikaryophase. The two nuclei in it eventually fuse. This is karyogamy. Karyogamy is equivalent to fertilisation. With karyogamy, the dikaryophase in the life cycle ends and the diplophase begins.
The septate ascogonium, ascogenous hyphae fusion or diploid nucleus (synkaryon) is the young ascus. It is the only diploid structure in the life cycle and thus represents the short-lived, transitory diplophase.
(iv) Differentiation of Ascospores Fig. 10.16 (F-I):
The young ascus enlarges. Its— diploid nucleus undergoes three successive divisions. The first and the second divisions constitute meiosis. The third is mitotic. The resultant 8 nuclei are thus haploid. A small amount of cytoplasm gathers around each daughter nucleus.
The uninucleate protoplasts secrete their own walls and are fashioned into uninucleate ascospores. Eight ascospores are thus formed by the method of free cell formation in each mature ascus which may be spherical or pear-shaped in appearance.
(v) Formation of Ascocarp (Fig. 10.17):
With the septation of ascogonium and development of ascogenous hyphae, a large number of sterile hyphae grow up around the sexual apparatus. The ensheathing sterile hyphae get interwoven to form a hollow ball- like structure, the peridium which surrounds and protects the ascogenous hyphae as they grow and branch within.
This is the ascocarp. The asci within the ascocarp are scattered. The ascocarp of Talaromyces is of indefinite growth. It continues to increase in size even after the after the ascospores begin to mature.
The peridium or sheath is thicker than that of the ascocarp of Aspergillus and consists of loosely interwoven hyphae. This entire structure is spherical and has no opening (A). Such a closed fruit or ascocarp is called the cleistothecium. The asci are borne in chains within the ascocarp.
In some other sp. of Penicillium, the peridium is compact and pseudoparenchymatous. The asci are borne singly and terminally on one-celled lateral branches of the ascogenous hyphae within it.
The cleistothecium of Penicillium or Aspergillus represents the following three generations:
(i) Parent haplophase:
It is represented by the sheath or peridium made up of loosely interwoven hyphae.
It consists of the binucleate cells of the ascogonium, ascogenous hyphae and the ascus mother cells. The transitory diplophase is represented by the young ascus containing a diploid nucleus.
(iii) Future haplophase:
It is represented by the ascospores in the asci.
Discharge of Ascospores:
At maturity the walls of the asci dissolve. The liberated ascospores, each shaped like a pulley wheel (side view), float in the nourishing fluids, formed by the degeneration of the inner layers of the peridium, walls of asci and the ascogenous hyphae. The ascospores absorb nutrition and mature. The mature ascospores are finally released by the decay of the other wall of the peridium.
Ascospore Structure (Fig. 10.18):
The liberated ascospore is a haploid uninucleate structure. It is lens-shaped with a small groove around the edge and thus appears like a pulley wheel in side view (B). The spore wall may be smooth or sculptured. It is differentiated into two layers, the outer epispore and inner endospore. The epispore is usually thick and sculptured. In face view the ascospore is round to star-shaped (A).
Alteration of Generations in Penicillium Brefeldianum:
In this species the sexual apparatus (antheridia and ascogonia) is absent. The vegetative hyphae give rise to two similar protuberances. They are known as the copulation branches.-The two copulation branches coil around each other. Their tips come in contact. At the point of contact the walls between the tips dissolve. The contents of one migrate into the other to establish a dikaryon in the fusion cell.
This species of Penicillium provides an example of isogamous sexual reproduction. After the migration of the contents the copulation branches are surrounded by sterile hyphae. The latter arise from the adjacent cells of vegetative hyphae. These sterile hyphae form a hyphal knot. The latter subsequently assumes a plectenchymatous character.
After about a week the outer membrane of the hyphal knot thickens to form a hard outer covering. The fusion or dikaryotic cell, in the meantime, sends out certain outgrowths. They are unseptate and are known as the ascogenous hyphae.
The dikaryon in the dikaryotic cell undergoes conjugate division. A pair of nuclei passes into each ascogenous hypha. The ascogenous hyphae become divided into short and cylindrical binucleate cells. Some of the cells grow out into buds with spirally coiled tips.
From the convex surface of these spirally coiled tips there arise branches that curve upwards and coil. From these branches there again arise short multicellular branches. Each cell of these last formed branches becomes spherical. It is known as an ascus. Nothing is known about the nuclear fusions in the ascus. Each ascus contains eight ascospores.
The asci containing ascospores, the ascogenous hyphae and the surrounding sterile hyphae with their outer hardened rind constitute a fructification. It is closed and is called the cleistothecium. The walls of the asci degenerate and the ascospores are liberated in the centre of the cleistothecium.
Later the outer hard rind of the cleistothecium also breaks. As a result the ascospores are released. Each ascospore possesses a double membrane the outer exospore and the inner endospore.