In this article we will discuss about the life cycle of phytophthora with the help of suitable diagrams.
Mycelium of Phytophthora:
It is profusely branched and consists of aseptate, hyaline, profusely branched, coenocytic, moderately thick hyphae about 4-8µ in diameter. The hyphal wall is approximately 0.1µ thick. It is electron transparent except for a thin, electron dense outer surface. Glucans is the predominant material in the hyphal wall. Cellulose is a minority component or even lacking altogether.
Hunsley and Burnett (1970) reported that in the hyphal wall of P. infestans are present B-1, 3- and B-1, 6-glucans as well as cellulose proteins. The plasma membrane is distinct and is seen as a dark line at the periphery of the hyphal protoplasm. It is undulating and frequently forms lomasomes. It is appressed to the inner surface of the hyphal wall.
The cytoplasm contains scattered nuclei, dictyosomes, lomasomes, mitochondria, endoplasmic reticulum, ribosomes and many large vacuoles with lipid inclusions.
Scattered throughout the cytoplasm are the lipid bodies more numerous in the old hyphae. Septa remain suppressed in the vigorously growing hyphae. They, however, appear here and there in the old hyphae or in connection with the formation of reproductive organs. The fungus spreads through tissues of leaves, stems and tubers.
The fungal hyphae ramify in the intercellular spaces between the cells of the host tissue. These are called the intercellular hyphae. In addition there are hyphae that penetrate, traverse and eventually leave the cell. These are called the intracellular or transcellular hyphae. The transcellular hyphae emerge from the host cell into an intercellular space or directly into a neighbouring host cell.
The intercellular and transcellular hyphae have similar ultrastructure. Haustoria develop on the intercellular hyphae when the latter come in intimate contact with primary walls of the host mesophyll cells. The hyphal wall is tightly pressed against host cell wall in this region which is called the penetration site.
Coffey and Wilson (1983) found that prior to haustorial development a lobed hemispherical deposit of moderately electron-dense amorphous material termed the penetration matrix is laid in this region of incipient penetration.
The bulging hyphal wall is intact in this region. The fibrillar network of the host cell wall, however, is no longer seen. It is replaced by the penetration matrix which is bounded by the host plasma membrane next to which is a thin band of host cytoplasm.
An infection peg arises from the hyphal bulge. It makes its way into the host cell pushing in front the host cell plasma membrane and the host cytoplasm. Within the host cell the infection peg expands at its tip to form the haustorium.
Haustoria of Phytophthora:
The haustoria are variously shaped intracellular feeding structures. In P. infestans they are small globes, occasionally short, straight or curved pegs. There may be one or more haustoria in each host cell. The intracellular haustorium is connected with the intercellular hypha by a neck-like constriction at the penetration site. Thus the long haustorial stalk or neck usually associated with this organ is lacking.
The electron-transparent haustorial wall is continuous with that of the intercellular hypha and so is the haustorial plasma membrane which encloses the anucleate haustorial cytoplasm containing a few mitochondria, lipid bodies and sparse endoplasmic reticulum.
Surrounding the haustorium, in close association with the haustorial wall, is a sheath of electro-dense amorphous material. It is termed the encapsulation zone or extra haustorial matrix (= sheath matrix).
It separates the haustorial wall from the host cytoplasm and thus forms the pathogen host interface between the fungal parasite and the host plant. The nature, origin and function of sheath matrix is still uncertain.
The extra haustorial sheath is delimited from the host cytoplasm by a tightly pressed unit membrane termed the extra haustorial membrane. The latter is considered the invaginated host plasma membrane. Ehrlich and Ehrlick (1966) did not find any infolding’s or deposition of host wall material surrounding the basal portion of the haustorium.
Hohl and Stossel (1976), however, found some haustoria covered by an additional layer of electron-transparent wall material between the extra-haustorial matrix and the extra-haustorial membrane. It is termed the wall apposition (A). The wall apposition is continuous with the host cell wall at its base. It may partially or completely surround the haustorium.
In the former case it forms a collar like structure at the base of the haustorium. Hohl and Sutter (1976) supporting Hohl and Stossel (1976) observed that wall apposition may or may not be formed. Coffey and Wilson (1983) reported that at an early stage the haustoria do not possess collars or encasements.
The haustoria are more commonly found in the tubers. In severe cases of infection the entire plant above the ground is killed. The fungus passes winter in the form of mycelium in infected potato tubers.
Asexual Reproduction in Phytophthora:
With the onset of favourable conditions of warm, damp weather in spring a tuft of slender, branching hyphae arise from the internal mycelium. They push their way out either through a stoma (A) or by piercing through the epidermal cell on the lower surface of the leaf. In the case of the tubers they push their way through the lenticels or injured portions of the skin.
These aerial hyphae are hyaline, and sympodially branched and are of indeterminate growth. These special branched aerial hyphae are called sporangiophores (conidiophores). Phytophythom, thus, differs from other members of the family in having sporangiophores distinct from the somatic hyphae.
The sporangium is formed by the inflation of the tip of the side branch of the sporangiophore (B1). The multinucleate inflation is then cut off by a transverse septum basal plug as a sporangium. The hyphal branch bearing the young terminal sporangium continues to grow (B2-3). As the sporangium reaches maturity, the branch tip swells slightly just below the sporangium and proliferates pushing the sporangium to the side as the elongation proceeds (B4).
The process may be repeated. The sporangia are, thus, borne terminally but are subsequently shifted to a lateral position. The mature sporangia are lightly attached, the sporangiophore of Phytophthora is, therefore, sympodially branched.
It bears nodular swellings (B5) which denote the points of detachment of sporangia. These swellings give the branched sporangiophore a jointed appearance characteristic of the genus.
Conditions governing sporangial production:
According to Crosier (1934) temperature and humidity govern sporangial production. The optimum temperature range is 18º to 22º regards relative humidity 100 per cent is the optimum and 91% mm— Cohen et al (1975) reported that sporangia are formed in abundance on infected leaves kept in darkness.
Under continuous light conditions no sporangia are produced by the fungus. He found that the inhibiting effect of light was temperature dependent. High temperature increased it. At 10º C 15 ºC, about 80% and 50% sporangia were produced in light whereas only 2-7% were produced at 20-25 C.
The mature sporangium is a hyaline, oval to elliptical, thin-walled spore sac with a basal plug. It has a small stalk and an apical papilla. The sporangial wall is nearly 0.3µ thick and appears to be faintly layered. Within the wall is the plasma membrane.
It is closely appressed to the sporangial well and encloses the multinucleate cytoplasm which contains the usual cell organelles such as mitochondria, dictyosomes, endoplasmic reticulum and ribosomes.
In addition the cytoplasm of the mature sporangium contains Paired basal bodies associated with the nuclei membrane bound packets of microtubules and several types of vacuoles. The chief among these latter are the cleavage and flagellar vacuoles.
The former which are considered to originate from the dictyosomes are randomly distributed in the cytoplasm. The flagella containing vacuolese are arranged along the sporangial wall. The mature lemon-shaped, pappilate sporangium with a basal plug is readily detachable.
Dispersal of sporangia:
Wind, rain splashes or contact with other leaves detach and spread or scatter the ripe sporangia on to the leaves of other potato plants. They may also be washed into the soil The sporangia lose their viability if they fail to germinate within a few hours.
Landing on the healthy leaf of a host plant, the sporangia germinate. The main factors governing germination are moisture and temperature. The sporangia are adversely affected by desiccation. According to Cochrane, they perish if the relative humidity falls much below 100 per cent.
In the presence of moisture provided by rain or dew, the sporaniga of phytophora infestants germinate either indirectly by producing zoospores and thus functioning as zoosporangia or directly by a germ tube and functioning as conidia.
The ability to germinate either directly or indirectly is influenced by temperature. Low temperature (12 to 15º C) induces zoospore formation and high temperature (20 to 23°C) favours direct germination by a germ tube.
The sporangia lose their vitality to germinate indirectly with increasing age. Young sporangia commonly reproduce by zoospores more readily than old sporangia. According to EIsner, Vande Molen, Horton and Bowen (1970) the loss occurs slowly at low temperature but more rapidly at higher temperature or following a heat shock of 40°C for 7-10 minutes.
(a) Indirect germination (Fig. 6.30):
Wet, cool soil favours indirect germination of sporangia. Thus in the presence of moisture at 16°C or below 12-14°C the sporangium behaves as a zoosporangium (A). According to Hohl and Hama moto (1967) the cleavage –cleavage vesicles which are randomly distributed in the cytoplasm become arranged radially equidistant from the neighbouring nuclei approximating the future planes of cytokinesis.
The flagellar vacuoles are arranged along the sporangial wall axially enjoined to the paired basal bodies. At the time of differentiation of zoospores, the margins of flagellar vacuoles fuse with the coalescing cleavage vacuoles.
The membranes of this system eventually fuse with the plasma membrane delimiting the multinucleate sporangial protoplast into 5-8 uninucleate daughter protoplasts (B) and freeing the flagella which Lie between the plasma membrane and sporangial wall. Each subsequently metamorphoses into a biflagellate zoospore which is reniform.
Of the two flagella one is of whiplash type and the other tinsel. Ferus (1954) reported the development of paddle-like structures at their tips. The flagella arise from the depression on the concave side. The zoospores are set free in a group by the bursting of the apical papilla into a vesicle in P. cactorum No vesicle is formed in P. infestans (C).
Indirect germination of sporangium through the intervention of zoospores is an example of the retention of an ancestral character. Phytophthora is a terrestrial fungus. Reproduction by zoospores is a primitive feature in its life cycle. Direct germination is considered an evolutionary adaptation.
The sporangia which are washed into the soil germinate and infect the tubers. As a result the tubers may rot by harvest time or rot during storage. Under favourable conditions a number of asexual generations may be produced in one growing season. This results in rapid propagation of the pathogen and spread of the disease at an amazing speed causing sudden and disastrous epidemic.
Germination of zoospore and infection of the host (Fig. 6.30 D-G):
The sporangia which land on the leaves of potato plants germinate in cool wet environment liberating zoospores. The liberated zoospores (D) swim about actively in a surface film of water for some time and then come to rest. Each quiescent zoospore retracts its flagella and may encyst (E). The encysted zoospore then germinates by putting out a special, short hypha called the germ tube (F).
Pristou and Gallegly (1954) reported the formation of an appressorium at the time of infection. The germ tube, as it grows over the epidermis of the leaf and closely adheres to it produces a flattened pressing organ, the appressorium at its tip (G2).
From the undersurface of the latter arises a fine tubular, peg-like outgrowth, the infection hypha, which pierces the cuticle and forces its way through the epidermis into the host tissue to bring about infection. The infection peg then swells to normal hyphal diameter.
The concept of penetration of foliage leaf direct through the epidermal cell, as suggested by Pristou and Gallegly (1954) was later confirmed by Shimony and Friend, (1975) and Wilson and Coffey (1970). Hohl and Suter (1976), however, reported that the principal method of entry into the host leaf is via stomata.
Direct penetration through the epidermis is not common. It is rather rare. After penetration via stomata a plug is often recognisable in the hypha at the level where it enters the leaf. No appressorium is formed in the case of entry through the stoma. Infection is high in cool, wet environment.
Within the host the invading hypha grows vigorously and branches to form the mycelium which is coenocytic and consists of intracellular and intercellula hyphae. In 1983 Coffey and Wilson found direct penetration of the host epidermal cells and the formation of apparessoria as the common method of infection. The infection peg splits the outer epidermal walls.
After penetration it swells to form the infection vesicle in the epidermal cell. The infection vesicle subsequently narrows down to form in intracellular infection hypha which grows into the underlying palisade cell. It grows downwards and finally leaves in to enter, intercellular space between the mesophyll (spongy) tissue.
Galactinase is the enzyme produced by P. infestans. It might aid the penetration of the tuber tissue.
(b) Direct Germination of Sporangium (Fig. 6.31):
In wet warm environment (at higher temperature from 20 to 23°C), the sporangium (1) behaves like a conidium and germinates directly by a germ tube (2). Harvey (1954) reported that at 25°C in water about 50 percent sporangia germinated directly while none produced zoospores.
In the India stages of direct germination vacuoles containing flagella and cleavage vesicles are formed as in indirect germination. Later, however, the flagella degenerate and cleavage of cytoplasm does not occur.
Instead a new wall layer is formed between the plasma membrane and the sporangial wall. It is termed the germination wall. In P. infestans, the germination wall is most conspicuous in the apical region of the germ tube but tapers away to nothing in the body of the sporangium.
Hemmes and Hohl (1969) reported that in P. parasitica the germination wall completely surrounds the sporangial protoplast and is continuous with the wall of the germ tube. Thus according to them the wall of the germ tube represents an extension of the germination wall. The germ tube pushes out through the central part of the apical plug area or the sporangial walls.
Beyond the plug area, the germ tube swells to form a vesicle or bulge. Form this vesicle arise one or more hyphal tips which continue the growth. Sometimes the emerging germ tube may branch directly without forming a bulge or may not branch at all. Most of the growth takes place at the hyphal tip.
Various species of Phytophthora produce chlamydospores. These develop as oblong intercalary or terminal spores. Most have relatively thick walls but this varies with the species. In a mature chlamydospore the cytoplasm is dominated by lipid and reserve vacuoles.
Sexual Reproduction in Phytophthora:
In Phytophthora sexual reproduction is oogamous. Till recently there has been some confusion with regard to the nature of sexuality in the different species of the genus. It has, therefore, been the subject of numerous investigations.
Some species were considered homothallic while others proved to be heterothallic. The former produce sex organs in a single culture but the latter require the presence of two compatible isolates (strains) for oospore formation.
The isolates of heterothallic species may be unisexual or potentially bisexual but produce sex organs under normal growing conditions only when two strains of opposite mating types interact.
There is no significant morphological difference between the two. They are potentially bisexual but self-sterile. Gallindo and Gallegly (1960), Savage et al. (1968) and Brasieur (1972) corroborated the view that the heterothallic species generally are bisexual.
From the results of various investigators Shepherd (1978) concluded that all species of Phytophthora are monoecious. It means their mycelium is potentially capable of producing both antheridia and oogonia. These monoecious species, however, differ in their mating capabilities. Thus, on the basis of their mating competence.
Shepherd (1978) divides them into following three categories:
(i) Self-compatible monoecists (homothallic species). In this category are included the self- fertile species.
(ii) Self-incompatible monoecists (Heterothallic species). These species produce sex organs only when two strains of opposite mating types (termed A1, and A2) interact. They are self-sterile.
(iii) Neuter or Sterile species. The strains which do not produce sex organs under normal conditions are included in this category.
According to the above classification, Phytophthora infestans is considered a self-incompatible monoecist (heterothallic). The isolates are potentially bisexual and morphologically alike.
Each strain produces both types of sex organs when the two strains of opposite mating types occur in the same host near each other. Gallegly and Gallindo (1958) used the symbols A1 and A2 to designate them. It is worthwhile to note that the mycelium of each strain in P. infestans produces both antheridia and oogonia which are self-sterile.
Mating, however, takes place between the sex organs of opposite mating types.
Oospores in this species are thus formed only if antheridium of A1 strain copulates with oogonium of A2 mating type and vice versa.
Antheridial incept (Fig. 6.32):
It arises as a short lateral hypha from the mycelium with its tip inflated to form a more or less clavate structure known as the young antheridium (A). It is thin-walled and contains non-vacuolated cytoplasm with a single nuclei before the entry of oogonial incept (A). Usually the young antheridium is not separated by a septum from the supporting hypha or stalk.
Oogonial incept (Fig. 6.32):
It also arises as a short, lateral hypha without any inflation (B). It contains dense, multinucleate cytoplasm.
The antheridial and oogonial incepts of the opposite mating types (A1 and A2) grow and curve towards each other. Eventually the tip of the oogonial incept of one strain comes in contact with the young antheridium of the opposite mating strain, punctures and grows through it (B) to emerge on the other side (C) where it swells into a globose structure, the oogonium (C).
The antheridium, now forms a collar-like structure surrounding the base of the oogonium (C). This type of antheridium is termed amphigynous.
Nature of amphigyny:
Two hypotheses have been put forth to explain amphigynous condition of antheridium in Phytophthora. Pethybridge (1914) reported that antheridial and oogonial hyphae (of opposite mating types. A1 and A2) grow towards each other.
The tip of the oogonial incept of one strain comes in contact with the developing antheridium of the opposite mating strain, penetrates it and grows through it without loss of contents (plasmogamy) and breaks out at the top to swell into a spherical or pear-shaped oogonium.
The antheridium thus appears sitting like a funnel- shaped collar at the base of oogonium. This view which has come to be known as the classical concept was confirmed by Mundkar (1949) using light microscopy, Vujicick (1971) using electron microscopy and many others.
Rosa and Lidegren (1925) and Cooper and Porter (1928), however, pointed out, that this interpretation of amphigyny is an illusion created by the wrapping of the antheridial initial around the oogonial stalk.
Stephenson and Irwin (1972) supporting Rosa and Lidergen reported that in P. capsici, the antheridial initial encircles the developing oogonial stalk and is not penetrated by the oogonial incept as classically described.
Hemmes and Bartinicki-Garcia (1975) using electron microscope reconfirmed the classical concept of amphigyny of antheridium in P. capsici.
(i) Contact between oogonial and antheridial hyphae,
(ii) Penetration of antheridial hypha by the oogonial initial,
(iii) Re-emergence of the oogonial initial, and
(iv) Oogonium expansion at the tip after emergence.
Ho and Zentmyer (1977) advocating the classical concept stated that in P. cinnamoni the amphigynous antheridia result from the penetration of antheridial initial by the oogonial initial.
The vertical juncture line demonstrated in P. capsici by Stephensen and Irwin (1972) to indicate the encirclement of oogonial stalk by the amphigynous antheridium was never observed in by ‘ Phytophthora’capsici blight either light or scanning electron microscopy. It is evident from the above that there is overwhelming evidence in favour of the classical concept of amphigynous condition of antheridium in Phytophythora.
Mature Oogonium (Fig. 6.32):
It lies above the antheridium and is spherical or pear-shaped in form (B). It has a dense, multinucleate cytoplasm when young (C). Towards maturity it increases in size. The protoplast becomes vacuolated. The nuclei which are spherical and about 40 in number undergo division which according to some workers is haploid mitotic and according to others meiotic.
At this stage the oogonial protoplast becomes differentiated into an outer or peripheral, hyaline zone with vacuolate multinucleate cytoplasm surrounding a central, uninucleate region with denser cytoplasm. The former is called periplasm and the latter ooplasm (D).
All the nuclei in the periplasm later disintegrate (E). Prior to fertilization the single nucleus in the ooplasm divides into two daughter nuclei (E). One of these denegerates. The surviving one functions as the egg or oosphere nucleus.
It is funnel-shaped, sits on the oogonial stalk forming a collar-like structure around the base of the oogonium. This type of antheridium surrounding the base of the oogonium is termed amphigynous. It is delimited from its urpporting hypha by a septum. The antheridium contains about 12 nuclei.
These are formed from the original two nuclei of the young antheridium, after penetration by the oogonial branch by division. The division according to some investigators is haploid mitotic and according to others meiotic. Prior to fertilization all the nuclei except one degenerate. The surviving one functions as the male nucleus.
Fertilization in Phytophthora:
The oogonial wall bulges at one point into the antheridium. This point is called the receptive spot. It then dissolves at the receptive spot. Through the opening the antheridium pushes the fertilization tube. Gallegly (1968) reported that in P. infestans fertilization tube is formed from the antheridium just above the cross wall of the oogonial stalk. It extends upward into the oogonium.
This was confirmed by Laviola (1974) who found that after its origin from the antheridium it penetrated the intervening walls between the antheridium and the oogonial stalk at the point of contact. Within the oogonium it travelled up the oogonial stalk, penetrated the and entered the oosphere at the nearest point.
Here it opens at the tip to deliver the male nucleus and some of the protoplasm. Sometimes the fertilization between the oogonial wall and the oosphere and entered the latter at nearly the region. In case the antheridium is bicellular the fertilization tube arise from the upper or distalcell above the septum or plug of the oogonial stalk.
Oospores have also been reported to be formed parthenogenetically.
The fertilized egg secretes a heavy wall around it and becomes an empty space between the oogonial wall and the thick oospore wall. The oospore thus partially fills the oogonial cavity and is described as aplerotic.
It is a resting spore and thus considered to be an important overwintering structure which plays a significant role as an important propagule in the disease cycle. In cultures sterols are necessary for the formation of oospores.
Zentmyer and Irwin (1970) reported that the fully dormant oospore has a thick wall differentiated into two or three Layers. There is a large, dense, spherical body in the centre with sharply defined edges known as the reserve gobule. The cytoplasm surrounding the central body contains several spherical globules perhaps lipid or oil in nature.
The male and female nuclei have been reported to fuse very letter during maturation of oospore. Backwell (1939) associated oospore dormancy the nature of thick oospore wall. It provides protection and is practically impermeable Germination cannot take place until it is rendered permeable to oxygen and water.
The oil or lipid globules provide energy for its long dormancy. Sussman (1966) and Schmitheshner (1968) reported that the democracy in Phytophthora oospore is probably constitutive due to the innate property of the dormant stage.
Furthermore, the reported absence of ribosomes and of cytochromes in the oospores of P. capsici shows that the metabolic activities of the mature oospore are at very low level.
Germination of oospore:
It increased with age. Romero and Irwin (1969) obtained 64% oospore germination when oospores of P. infestans were kept at 4°C for 45 days. Germination takes place after the decay of the host tissues and on the onset of conditions suitable for germination, Gough et al reported that germination of oospores of P. infestans occurred at temperatures raging from 12°C to 25°C.
During the pregermination stage Phytophthora oospore absorbs water and swell. The diploid oospore nucleus divides repeatedly. The increase in the number of nuclei is accompanied by increase in the number of mitochondria.
The central globule by vacuoles indicating its digestion and absorption. The pitting and erosion of the thicker layer which becomes thin at the site of germ tube emergence is indicative of impending germination. At this stage the oospore protoplast covered by the thinned inner layer bulges at the site of emergence and makes its way out through the outer layer of the oospore wall and presses against the oogonial wall.
Finally it breaks through the proximal end of the oogonial envelope or emerges by reputuring the oogonial wall at other locations. Growth of the germ tube after emergence was variable. In P. cactorium directly grows into a mycelium.
The germ tube in P. infestant usually of ends in a terminal papillate germ sporangium typical of the species in methods of germination. The contents of the terminal sporangium may divide to form zoospores. Each zoospore (J) after liberation germinates to give rise to the mycelium. The terminal sporangium may also germinate directly by forming a germ tube.
Life Cycle and Location of Meiosis in Phytophthora:
Till recently the widely held view was that Phytophthora has a haploid life cycle (6.33A). The only diploid structure in the life cycle is the oospore. Meiosis takes place in the zygote. The scheme of zygotic meiosis and haploid cycle in Phytophthora has been reconfirmed by Timmer et al. (1970), Zentmyer and Irwin (1970) and Stephens et al. in P capsici. Sansome (1965) sounded a discordant note.
She suggested gametangial meiosis in Phytophthora. On this basis the haploid phase in this fungus is extremely reduced and restricted to the gametes only. Further evidence in favour of diploid life cycle and gametangial meiosis (Fig. 6.33 B) was furnished by Sansome (1976) in P. capsici.
She observed pachytene, diplotene and diakinesis stages characteristic of meiosis I in the oogonium thus confirming gametangial meiosis in the life cycle of Phytophthora.