In this article we will discuss about the life cycle of plasmodiophora brassicae with the help of suitable diagrams.
Cook and Swartz (1930) showed that the life cycle of P. brassicae comprises two distinct phases, the haplophase (primary phase) and the diplophase (secondary phase). The former occurs in the root hairs and the latter in the cells of the main root cortex of the susceptible host plant.
Each phase includes a number of successive stages which are described below:
The resting spores or sporangia (cysts) which are differentiated by meiosis from the diploid protoplast of the sporothallus (secondary Plasmodium) are pioneer structures of the haplophase which occurs in the root hairs.
1. Fine Structure of Resting spore or sporangium (Fig. 5.2):
It is a tiny hyaline, spherical, uninucleate, haploid structure up to 4.0 µ m in dia. According to Williams and Menabola (1967) the sporangial wall is differentiated into 3 layers (A) but in many cases it showed only 2 layers.
The outer layer is electron-dense and granular. It included the surface spinules. The middle layer is less electron-dense. Yukawa and Tanaka (1979) described a circular germ pore in the sporangial wall with the middle layer (inner in the 2 layered sporangial wall) thickened to form a circular electro-lucent plug-like structure in the region of the pore orifice (B).
It is surrounded by a ring of electron-dense material contiguous with the inner wall layer. Within the sporangial wall is the sporangial membrane which completely invests the cytoplasm replete with ribosomes. The other organelles present in the cytoplasm are mitochondria, endoplasmic reticulum and dictyosomes.
It has a single haploid nucleus. Numerous lipid globules of various sizes is another conspicuous feature of the spore (sporangial) cytoplasm. The mature spores are set free, when the clubs undergo decay. In the soil they germinate to initiate the life cycle.
2. Germination of Resting Sporangium or Spore (cyst) (Fig. 5.3):
The spores of P. brassicae may germinate immediately or remain dormant in the soil for several years.
(a) Conditions affecting germination:
According to Hofing (1930), the most important factor for germination is maturity. Thus the spores or sporangia from older decaying galls germinate faster than those from the young galls. Ellison (1946) noted mutual stimulation of germination by resting sporangia (spores) in masses. Bremer (1924) reported that germination occurred more readily in wet acid than in limed soils.
The early suggestion of Chupp (1917) that the presence of host seedling stimulates germination, under certain conditions, has been substantiated by Macfarlane (1959, 67) and Bochow (1965) for P. brassicae. According to Macfarlane (1970) presence of host or metabolites secreted by them promote germination.
(b) Process of germination:
Chupp (1917) and other early workers reported that the spore (A, sporangium) germinated in a favourable medium immediately. It absorbs water and swells considerably (B), particularly on one side along which a slit appears in the spore or sporangial wall. Through the slit, the uninucleate protoplasm emerges (C) as a single swarm cell or zoospore which is spherical or pear-shaped (D) in form.
Tommercup and Ingram (1971) observed a single, uninucleate, naked protoplast emerging from an isolated point on the spore wall. Macfarlane (1970) and Aist and Williams (1971) using interference optics observed a germination pore in the resting sporangium (or spore) wall through which the protoplast emerges slowly.
Recently Yukawa and Tanaka (1979) observed a circular germ pore in the sporangial or spore wall with a thickened plug-like structure (Fig. 5.2 B) in the pore orifice. In their view, this region undoubtedly constitutes the region of the germination pore of the resting sporangium (or spore) seen by Macfarlane and Aist and Williams.
On germination, the plug dissolves to enable the zoospore to emerge through the germ pore. The refractile (lipid) globules in the sporangial cytoplasm gradually disappear as the germination begins. The spore contents become less viscous.
A small papilla emerges through the germ pore in the spore wall. It enlarges to form a thin vesicle as the spore contents flow into it. The flagella develop on the spherical or pearshaped zoospore as it completely emerges. It may also be termed as the primary or cyst zoospore.
Primary or Cyst Zoospore (D):
It is naked and extremely active after emergence for several minutes. Uptill 1934, it was assumed that the zoospore is furnished with a single flagellum inserted at the anterior end. It is of whiplash type and several times longer than the length of the zoospore. Ledinghan (1934) observed zoospores furnished with two unequal flagella.
Ellison (1945) confirmed that the zoospores of P. brassicae are biflagellate. Kole and Geilink (1962) observed with electron microscope that each zoospore is furnished with unequal, whiplash type flagella at its anterior end—a long one with an end piece and a short one arising by its side with a blunt end. The latter lacks the end piece.
3. Primary Infection of Host (Fig. 5.4.):
Infection takes place at the seedling stage in the host. The biflagellate, heterokont (with unequal flagella) primary zoospore (cyst zoospore) swims in a film of water in the soil or creeps over soil particles like an amoeba.
According to Ayers (1944), on contact with the root hair of suitable host seedling (Cabbage or other crucifer seedling), the zoospore becomes attached and amoeboid, in this state it is called myxamoeba (A1).
The myxamoeba retracts its flagella and enters the root hair directly by a partial dissolution of the root hair wall (A2). According to Aist and Williams (1971), there is rapid injection of the parasite (encysted cyst zoospored) into the root hairs of the host by means of the special injection apparatus developed in the encysted zoospore.
The cyclosis of host cytoplasm sweeps the myxamoebae away from the penetration site soon after injection. The young myxamoebae in the root hairs are separated from the host cytoplasm by two kinds of simultaneously occurring intrafacial membrane arrangements. One of these is a 7-layered envelope of two closely appressed unit membranes and the second host parasite interface is also a 7 layered envelope.
4. Primary Plasmodium (Gametothallus, Fig. 5.4 A):
Within the root hair the uninucleate myxamoeba (A2) enlarges to form a thallus. The protoplasm increases in size and the haploid nucleus divides repeatedly. The divisions are cruciform. The resultant small naked, multinucleate acellular mass of protoplasm constituting the thallus is called the primary Plasmodium.
It is a haploid structure and thus termed gametothallus. Many such thalli may be present in one root hair, if multiple penetration occurs, but these do not fuse.
5. Sexual Reproduction. (Fig. 5.4 B-C):
The haploid primary Plasmodium (gametothallus) is concerned with sexual reproduction. After a certain period of growth, the cytoplasm of the gametothallus, while still lodged in the root hair, cleaves into as many uninucleate daughter protoplasts as there are nuclei (usually 4-10) in the Plasmodium (B4).
Each daughter protoplast secretes a membrane around it and functions as a gametangium. The gametangia are small spherical, thin-walled uninucleate structures. The haploid nucleus of the gametangium undergoes mitosis to form four to eight daughter nuclei (B5).
Each daughter nucleus gathers a small bit of cytoplasm around it and develops into a spindle-shaped motile, biflagellate, isogamete which is smaller in size than the zoospores (C6). The two flagella are of unequal size and of whiplash type inserted at the anterior end. Thus in one gametangium up to eight isogametes are formed (B5).
They have no cell walls. The whole process takes 2—8 days. The thin gametangium wall bursts (C6) and liberates the gametes in the root hair. All the stages (A—D) described above occur in the root hairs of the host. These constitute the haplophase.
Plasmogamy (Fig. 5.4 B6, C7):
After liberation, the isogametes copulate in pairs. It has not been established whether gametes from the same gametangium or from different gametangia copulate. It is also not known definitely whether pairing of isogametes takes place in the root hairs or cortical cells after migration.
The most widely held view is that the isogametes are released in the soil outside the host through a pore formed at the point of contact of gametangium and the root hair cell wall. Pairing of gametes takes place outside the host in the soil. Pairing of gametes is soon followed by the fusion of their membranes and cytoplasms. This is termed plasmogamy. The latter thus results in the formation of binucleate fusion cell (D8).
The fusion of two nuclei in the binucleate cell which is termed karyogamy is delayed. It takes place immediately before cyst formation.
This phase in the life cycle begins with the formation of quadriflagellate zygotes (Dg). The single nucleus in zygote is diploid.
6. Zygote (Fig. 5.4D):
The quadriflagellate zygotes are generally found in the cortical cells of the root. As mentioned above it is not definitely known whether the gametes fuse in the root hairs or in the cortical cells after migration. In the former case the flagellated zygotes perforate the epiblema cell walls and reach the meristematic zone of the root. When the quadriflagellate zygote is formed in the soil, it reinfects the root.
Once in the root cortex, the zygotes retract their flagella and become amoeboid. The amoeboid zygotes are called myxamoebae (E9). The myxamoebae spread from cell to cell by direct penetration of cell walls. At first they are found in the cortical cells and later in the other tissues of host root and stem.
7. Secondary Plasmodium (Sporothallus, Fig. 5.4 F10):
Within the invaded cortical cell, the myxamoeba by subsequent growth accompanied by repeated karyokinesis (mitosis) of its diploid nucleus but no cytokinesis, grows to form a relatively large, naked, multinucleate diploid Plasmodium.
It is called the secondary Plasmodium or sporothallus or cystogenous Plasmodium. In the mature state, it may occupy the entire lumen of the infected host cell and surround the host cell nucleus (Fig. 5.5 A). It lies immersed in the cytoplasm. The host cell may contain more than one plasmodia.
Ultrastructure of Secondary Plasmodium (Fig. 5.6):
It was investigated by Willams and McNabola (1967). The plasmodium is bounded and thus separated from the host cytoplasm by a smooth, deeply staining osmophilic membranous envelope about 250A thick. It consists of two closely appressed osmophilic membranes. There is no cell wall within the membranous envelope; the growing plasmodial cytoplasm is replete with free ribosomes.
Besides, it contains abundant endoplasmic reticulum and several mitochondria, each with a few tubular cristae. Dictyosomes occur near the diploid plasmodial nuclei. The presence of numerous prominent lipid droplets of variable sizes is another conspicuous feature.
Each nucleus of the multinucleate plasmodial cytoplast is bounded by a perforated nuclear membrane and contains a single prominent nucleolus. A number of microtubules have been reported to occur near each diploid nucleus. The intracellular Plasmodium of P. brassicae lacks special feeding structures.
8. Formation of Clubs:
The presence of the secondary plasmodia in the cortical host cells elicits many metabolic responses in the infected cells. One of these is the excessive or abnormal increase in the size of the infected host cells. This is termed hypertrophy. Besides hypertrophy,’ the presence of secondary plasmodia in the cortical cells stimulates them to have active and repeated division.
This excessive multiplication of infected or diseased host cells is termed hyperplasia. Hypertrophy and hyperplasia of the infected cells stimulates the neighbouring uninfected parenchyma cells of the host to divide and redivide.
Consequently there is excessive proliferation of infected host cells in the form of tuberous swellings or malformations on the diseased roots. These malformations are called clubs or galls. This tumorous growth (galls or clubs) of roots and hypocotyl resulting from cell hypertrophy and hyperplasia is the most striking feature of Brassica plants infected with P. brassicae.
Williams, however, suggested that the stimulus for cell division and cell hypertrophy in the club root galls lies within the parasitized cells only and is not transferred to the adjacent cells. The infected roots act as sinks for growth materials produced in parts of the plant above the ground.
The club-tissue is thus likely to be invaded by soft rot bacteria or other rot producing organisms which bring about decay of clubs resulting in the formation of materials toxic to the plant and cause wilting of the tops. Subsequently it may result in permanent wilting or retarded growth.
9. Vegetative Multiplication of the Parasite (Fig. 5.5):
At the time of division of infected or diseased host cells (hyperplasia), the secondary plasmodia may also divide (B), a part of it going to each infected daughter cell. The formation of new plasmodia by a vegetative method of fragmentation results in the spread and propagation of the disease.
Groups of infected hypertrophied cells become recognisable in the host tissues (C). Woronin (1878) called each such group of hypertrophied cells Krankhaitsherd (C). The secondary Plasmodium of each enlarged cell of Krankhaitsherd eventually becomes converted into a mass of resting sporangia or cysts (E).
10. Differentiation of Resting sporangia (Cysts) or spores also called cyst (Sporulation stage Fig. 5.5 D-E):
The mature multinucleate diploid secondary Plasmodium (sporothallus) practically fills the entire lumen of the hypertrophied cortical cell (D). It ceases to grow in size. With the cessation of vegetative growth, a new phase in the life cycle ushers in. During this phase, the diploid nuclei of the Plasmodium seem to lose their contents and disappear.
The chromatin fails to take the usual stain. It is the akaryote stage. Soon after the nuclei reappear and the protoplasmic mass of the Plasmodium cleaves around each nucleus. The entire Plasmodium is thus transformed into a large number of tiny uninucleate, spherical bodies each of which is surrounded by a wall of its own. These are termed the resting sporangia (Cysts) or spores (E).
Till recently the nature of the akaryote stage was an enigma. Lutman (1913) suggested that meiosis takes place during sporangial (Cysts) or spore differentiation. Milovidon (1931) claimed that akaryote stage represents prophase I of meiosis. Garber and Aist (1979) conclusively showed that the akaryote stage represents the meiotic prophase I and the subsequent divisions are remainder of meiosis I and meiosis II.
They thus confirmed previous claims both of Lutman (1913) and of Milovidon (1931). According to Garber and Aist’s study, the akaryote stage marks the transition between the actually parasitic vegetative stage represented by the diploid Plasmodium and the dormant phase represented by the resting sporangia (Cysts) or spores which are haploid structures.
The vacuoles and long slender furrows which are absent in vegetative phase (secondary Plasmodium) appear in the plasmodial cytoplasm in the meiotic process. The vacuoles appear in the prophase I and furrows during metaphase I. The latter demarcate the boundary along which cleavage will occur.
Both the vacuoles and furrows become prominently conspicuous at the beginning of meiosis II. The plasmodium is still a single unit at this stage. By the end of meiosis II, the Plasmodium is divided into a number of tiny daughter protoplasts, each with 2 haploid nuclei.
The final step in cytoplasmic cleavage is the division and separation of the tiny binucleate blocks of plasmodial cytoplasm into uninucleate, irregularly-shaped haploid daughter protoplasts which later round up to take on the shape of mature haploid resting sporangia (Cysts) or spores.
Each young sporangium or spore is invested by a membranous envelope known as the sporangial membrane. The entire secondary plasmodium is thus converted into a sporangial or spore mass.
The spores or sporangia (Cysts) in the mass are free from one another but the entire mass of sporangia or spores is surrounded by the common plasma membrane of the host cell. A fine fibrillar matrix termed the inter-sporangial matrix remains between the sporangia or spores in the mass.
According to Williams and McNabola (1967), granular aggregates of the intersporangial matrix are deposited along the sporangial or spore membrane as small spines termed the spinules. Towards maturity, sporangial or spore wall is deposited between the sporangial or spore membrane and the spines.
Ultra-structurally, the cyst spore wall is formation of resting differentiated into 5 layers.
(i) An outer layer of loose proteinaceous fibres (l1),
(ii) A layer of similar fibres but with lipid granules enmeshed (l2),
(iii) A prominently chitinous wall (l3),
(iv) An inner phospholipid containing region (l4 ) and
(v) An inner spore membrane.
The chemical analysis of cyst wall shows that the principal components are chitin, protein and lipid. The mature sporangia of spores (Cysts) are set free in the soil when the club roots undergo decay and decomposition by soft rot micro-organisms.
The chitinous nature of the sporangial or spore (Cysts) wall enable them to withstand adverse conditions. The resting spores or cysts (sporangia) thus perennate in the soil.
They germinate to produce zoospores on the offset of suitable conditions to continue the fresh life cycle. Macfarlane (1970) reported that the spore or cyst germination is stimulated by the presence of susceptible host seedlings or by certain of their metabolities.
Summary of the Life Cycle of Plasmodiophora Brassicae:
It comprises two distinct generations or phases namely the gametophyte generation or haplophase and sporophyte generation or diplophase. The former occurs in the root hairs and the latter in the cortical cells of the host root (Brassica sp.).
The gametophyte generation (haplophase) is initiated by the differentiation of resting spores (sporangia) or cysts by meiosis from the diploid protoplast of the secondary plasmodium in the club roots of the host cortex. The mature cysts or resting sporangia or spores which are haploid are released into the soil when the club roots undergo decay and decomposition by soft rot microorganisms.
After liberation, they perennate or germinate immediately if the conditions for growth are favourable. On germination, each resting spore or cyst produces a biflagellate, heterokont, haploid zoospore.
The released zoospore swims in a thin film of water in the soil of creeps over the soil particles like an amoeba. Coming in contact with the root hairs of a suitable host it becomes attached and amoeboid. In this state, it is called a myxamoeba. It then retracts its flagella and enters the root hair.
Within the root hair it is swept away from the penetration site by host cytoplasmic cyclosis. By growth and repeated cruciform nuclear division, the uninucleate myxamoeba becomes converted into a naked multinucleate thallus like mass of protoplasm. It is acellular haploid and is called the primary plasmodium (or gametothallus).
Reaching a certain stage of maturity the haploid protoplast of gamete thallus cleaves into 4-10 small, spherical, thin-walled, uninucleate gametangia. The haploid nucleus of each gametangium undergoes repeated mitosis to form 4-8 nuclei. Each daughter nucleus gathers a bit of cytoplasm around it to become a spindle-shaped mobile biflagellate isogamete.
All the above mentioned haploid stages namely, the resting meiospores (sporangia or cysts), the zoospores, the primary plasmodium (gametothallus), the gametangia and the isogametes (which occur in the root hairs of the host) constitute the gametophyte generation or haplophase.
The second phase of the parasite life cycle commonly called as the diplophase or sporophyte generation starts with the formation of zygotes. The zygotes are found in the cortical cells of the root. It is not definitely known whether the isogametes fuse in the root hairs or in the cortical cells after migration.
The consensus favours the view that the isogametes are released in the soil outside the host where pairing followed by plasmogamy takes place. Karyogamy is delayed. A binucleated quadriflagellate zygote thus formed reinfects host root.
Within the root cortex, the zygote retracts its flagella and becomes amoeboid in form. It is called myxamoeba. Within the invaded cell the myxamoeba by subsequent growth accompanied by repeated cruciform division grows to form a naked, multinucleate diploid mass of protoplasm called the secondary plasmodium or sporothallus.
It fills the lumen of the cortical cell. It then ceases to grow in size and its diploid protoplast undergoes meiosis.
As a result, the multinucleate diploid protoplast of the secondary Plasmodium is converted into a large number of uninucleate spherical haploid bodies each of which is surrounded by a wall of its own. These are the resting spores (sporangia) or cysts.
The resting spores when mature are set free in the soil by the decay of club by rot microorganisms. In the soil the resting spores or cysts germinate under favourable conditions to continue the fresh life cycle. The zygote and the secondary plasmodium constitute the diplophase or sporophytic generation.
Alternation of Generations of Plasmodiophora Brassicae:
There is distinct alternation of generations in the life cycle of Plasmodiophora. The zygote and the secondary plasmodium (sporothallus before spore formation) represent the sporophyte generation or diplophase. The resting spores, the zoospores, the haploid primary plasmodium (gametothallus), the gametangia, and the isogametes represent gametophyte generation or haplophase.
The two crucial points in the life cycle are meiosis at the time of differentiation of spores (meiospores) and karyogamy (fusion of gametic nuclei). At these critical points, the life cycle switches on from one generation to the other.
With meiosis the diploid or sporophytic phase ends. The gametophyte phase starts. At the time of karyogamy, the gametophyte phase terminates and the sporophytic phase is initiated with the formation of diploid zygote.
These two generations occur regularly one after the other in the life cycle. This is called alternation of generations. The life cycle characterised by alternation of generations with sporogenic meiosis is called haploid-diploid or diplohaplontic (diplobiontic life cycle).