In this article we will discuss about the life cycle of pythium with the help of suitable diagrams.
Somatic Phase in Pythium:
The mycelium (Fig. 6.18 A) represents the vegetative or somatic phase of the fungus. It is well developed, and coenocytic. It presents a white, fluffy appearance and consists of long rather slender, hyaline much branched hyphae which are very much like the filaments of Vaucheria. Usually there is a slight constriction at the base of side branch.
The hyphae are generally regular in diameter but taper evenly towards their tips. The young vigorously growing hyphae are not divided into cells. The septa, however, appear in connection with the no distinction into rhizoidal and aerial hyphae.
It may live saprophytically or parasitically. Saprophytically it lives on dead organic matter in the soil. Parasitically it grows in the young seedling. The entire mycelium may be within the host tissues or some hyphae may lie within the host and others outside the host. The hyphae which lie within the host tissues are both intracellular and intercellular.
They do not produce haustoria. The external hyphae may infect fresh seedlings growing in the neighbourhood or bear sporangia (Fig. 6.18 B). At first the fungal hyphae are restricted to the cortical region of the hypocotyl.
Later they make their way into the vascular bundles and absorb food substances. The whole plant thus comes to be infected by the mycelium. The appropriation of food and production of waste materials by the fungus parasite result in the death of the host. Thereafter the parasite lives on the dead remains as a saprophyte.
Hyphal Structure of Pythium:
Generally it is held that the hyphal wall contains cellulose. According to Mitchell and Sabar (1966) and Bartnicki-Garcia (1966), cellulose is a minority component or even lacking altogether Monocha and Colvin (1968) also could not detect the presence of cellulose in the Phythium hyphal wall.
B glucans is the predominant material. Mature hyphal wall is differentiated into two distinct layers which differ in the arrangement of microfibrils.
The outer layer contains randomly dispersed micro-fibrils and the inner has longitudinal arrangement. The microfibrils give reflections different from cellulose I or chitin.
Within the hyphal wall is the plasma membrane which forms lomasomes. The plasma membrane encloses the hyaline cytoplasm which becomes vacuolate in the older plants. The hyphal cytoplasm contains numerous small randomly dispersed nuclei, mitochondria and dictyosomes.
The endoplasmic reticulum, and cytoplasm ribosome are abundant and in the young hyphae. The reserve food is in the form the glycogen and there oil globules in addition. In the presence of lomasomes and dictyosomes, Pythium resembles green algae more than any other member of Mycota.The hyphal growth is by apical extension.
Reproductive Phase in Pythium:
The reproductive phase sets in much before the death of the host seedling. Reproduction is both asexual and sexual.
Asexual Reproduction (Fig. 6.18 B-H):
It takes place by means of zoospores which are produced in small, globular or oval, sac-like sporangia. The sporangia are formed singly and terminally at the ends of somatic hyphae which project into the damp atmosphere from the mycelium within the host tissue (B). There are no specialized sporangiophores.
The sporangia measure 15- 26 microns in diameter. Sometimes the sporangia are intercalary (I) The high humidity in the air promotes the growth of the fungal mycelium and the production of zoospores. Each sporangium is a multinucleate structure.
It is separated from the rest of the hypha, which still retains the power of elongation, by means of a transverse septum. The sporangiam is pushed aside and the hyphal tip grows to form the second sporangium. The sporangium is filled with hyaline cytoplasm containing numerous nuclei. It is slightly denser than that of young somatic hyphae.
(a) Indirect Germination of Sporangia (Fig. 6.18):
Under wet conditions sporangia remain attached to the hyphae bearing them. They function as zoosporangia. The mature zoosporangium puts out from its side or at its apex a narrow, papilla-like outgrowth (C).
It is tubular at first and called the exit tube. The apex of the exit tube swells up into a thin-walled, tiny, balloon-like vesicle (D) The contents of the zoosporangium by now have divided into a small number of uninucleate, daughter protoplasts.
These migrate into the vesicle through the exit tube and become metamorphosed into zoospores. According to some, the undifferentiated protoplast of the zoosporangium migrates through the exit tube into the vesicle where differentiation of zoospores takes place.
The zoospores when mature, exhibit rocking motion and bounce on the vesicle wall. Consequently the vesicle suddenly bursts. The exit tube may often persist after the rupture of the vesicle.
The released zoospores swim and scatter in all directions (E). The tinsel flagellum is directed forward and the longer whiplash trails behind when the zoospore is in motion in the thin film of water on the host surface or in the soil.
Fine Structure of Zoospore (Fig. 6.19):
According to Lunney and Brand (1976), mature swimming biflagellate zoospore of Pythium proliferum is typically ovoid in form with a deep longitudinal groove. Viewed on end it presents reniform appearance. The two flagella are inserted laterally. They are attached to a distinct protuberance within the groove region. The anterior flagellum is of tinsel type and the posterior one of whiplash type.
The nucleus which is beaked in the freshly cleaved zoospore is now spherical and has a distinct eccentrically-located nuclelus. The dense cytoplasm is rich in ribosomes except the groove region which is ribosome free. There are several perinuclear dictyosomes which proliferate vesicles which fuse resulting in a reticulate, internal membrane system in the groove region.
Lunney and Brand (1976) termed it the “water expulsion vesicle”.
The nucleus is bounded by concentric bands of rough endoplasmic reticulum except towards the groove region where there is a closely appressed packet of ribosomes. Embedded in the cytoplasm are numerous mitochndria with tubular cristae and various types of vesicle inclusions such as the lipid vesicles, microbodies, cell vesicles and peripheral vesicles.
The rough endoplasmic reticulum cisternae ramify throughout the cytoplasm to the periphery.
The two kinetosomes of the flagella are located at the protuberance (in the groove region) adjacent to the nucleus. An electron opaque rootlet, which is striated, stretches between them. Two more fiber bundles originate from the kinetosome.
One of these runs to the anterior and the other to the posterior region of the zoospore along the plasma membrane. Microtubular roots radiate perpendicularly from the rootlets and are distributed into the different parts of the cytoplasm of the zoospore.
Encystment of Zoospores:
After swimming actively for a short or long period extending over several hours, depending on the environmental conditions, the zoospores show signs of fatigue. Their movements become slow and jerky.
Finally they become quiescent, retract their flagella and become spherical. Soon after the spherical resting zoospore secretes a delicate wall around it to become encysted. The encysted zoospore is usually called a cyst or cystospore (Fig. 6.18F).
Fine Structure of Cyst (Fig. 6.20 A):
The mature cyst is spherical in form (A). It has a smooth spore surface. Within the electron-opaque cyst wall is the plasma membrane. The cytoplasm contains a single eccentrically located nucleus with its perinuclear dictyosomes. The concentric layers of rough endoplasmic reticulum no longer surround the nucleus.
They appear to have been broken up into smaller rough endoplasmic reticulum packets which are evenly distributed in the cyst cytoplasm. The other cell organelles such as the mitochondria, ribosomes and vesicles are uniformly scattered in the cytoplasm.
Cyst Germination (Fig. 6.20 B):
Depending on the environmental conditions the germination of the cyst or cystospore may occur immediately or it may be delayed.
The condirions favouring germination are:
(i) Ill-drained and ill-aerated soils,
(ii) Moisture content of soil medium too high and
(iii) Temperatures also comparatively high. The cyst usually germinates by putting out a germ tube (G). Prior to germ tube emergence, the cyst nucleus undergoes mitosis once to form two daughter nuclei and the cell wall vesicles accumulate along the plasma membrane.
One of the daughter nuclei migrates into the germ tube. The latter finds its ways into fresh seedlings. The disease spreads rapidly by external hyphae and zoospores under conditions mentioned above.
Hohnk (1932) reported that the contents of the encysted zoospore may sometimes form a single secondary zoospore as in Saprolegnia and Achlya but it is of the original type. The zoospore of pythium the cyst and germinates to give rise to a fresh mycelium.
This behaviour of zoospores of Pythium must not be confused with the diplanetic condition of zoospores of Saprolesn. It is not diplanetism because the primary and secondary zoospores in Pythium are morphologically similar.
(b) Direct Germination of Sporangium (Fig. 6.18 G-H):
The sporangia get detached from the hyphae bearing them and are blown by the wind or dispersed by water. On falling on or reaching a suitable host the sporangium germinate like a conidium if the temperature is above the optimum. It directly put out a germ tube (G) which finds its way into the host either through a stoma or directly through an epidermal cell by secreting an enzyme at its tip.
Inside the host it grows and branches to form the mycelium (H). In this case the mycelium develops without the intervention of zoospores. Some mycologists call such sporangia which germinate directly and thus behave like conidia as conidiosporangia.
Others hold that these sporangia-like structure are in fact conidia. The production of conidiosporangia or conidia permits spread of the disease under relatively dry conditions.
Asexual reproduction in Pythium may also take place by means of gemmae. They are intercalary in position and globose in form. Occasionally the gemmae develop thick walls and are called the chlamydospores.
On germination the gemma or chlamydospore produces a long, tubular hypha which develops a sporangium at its free, distal end .The protoplast of the terminal sporangium produces zoospores.
Kinds of Sporangia:
In Pythium the sporangia are of two principal types, spheroidal or globose and elongate or filamentous.
(a) Globose sporangia:
They are more or less rounded in shape and are much broader in diameter than the hyphae and are usually terminal in position. The typical example of this category is P. debaryanum. Sporangial proliferation has been reported in the globular sporangia of P.prolifrum.
When the sporangium has shed the zoospores its base bulges up to form a new sporangium which matures within the primary empty sporangium or outside it. P intermedium also produces globular sporangia but in chains (I). They may function as zoosporangia or as conidia. The spherical sporangia of P. ultimum always function as conidia and germinate directly by producing a germ tube.
(b) Filamentous or elongate sporangia:
They are indistinguishable from the vegetative hyphae and have the same diameter as the hyphae bearing them. However, they have denser contents. The filamentous sporangia may be simple as in P. gracile and P. monospermum or branched as in P. aphanidermatum and P. myriotylum. In the latter case the sporangia are branched in a digitate manner and are slightly inflated.
Evolution of Conidium:
It is evident from the foregoing account that the different species of Pythium can be arranged in a series which shows a transition from a zoosporangium to a true conidium. The series starts with P. gracile in which the elongate sproangium exclusively produces zoospores.
Normally P. debaryanum also reproduces asexually by the formation of sporangia which in the damp atmosphere always function as zoosporangia. However, in the dry weather or when the temperature is above the optimum, the sporangium behaves like a conidium and germinates by a germ tube.
In P. intermedium the sporangia normally functions as zoosporangia as well as conidia.
P. vexans is an example in which the sporangia usually function as conidia and rarely as zoosporangia. The germ tube is initiated in place of the papilla of a zoosporangium.
In P. ultimum the sporangium invariably functions as a conidium and germinates by a germ tube. The zoosporangia are not produced at all.
The gradual transition of the sporangium to a conidium is an adaptation to a subaerial habitat.
Sexual Reproduction (Fig. 6.21 A-E):
It is oogamous and takes place by gametangial contact at the end of the growing season. At this time the food is nearly exhausted. There is not enough moisture for active growth. At this stage the host has been killed and the fungus is living saprophytically.
The sex organs are formed within the dead tissues of the host. The male sex organ IS called the antheridlum and the female oogonium. They are developed in close proximity on separate short, lateral hyphae arising from the same mycelium. Pythium debaryanum is thus homothallic.
Development of oogonium:
The tip of the female hypha inflates to form a globular swelling(A). Into the terminal swelling migrate the hyphal cytoplasmic contents. Finally the terminal swelling is cut off from the parent hypha by a basal hyphal plug [Haskins et al (1976)] or septum usually after, but sometimes before antheridial contact. The separated terminal swelling functions as the young oogonium.
Structure of young oogonium:
Ultrastructurally the young oogonium has a thin wall. Within the oogonial wall is the plasma membrane enclosing an electron-dense cytoplasm rich in endoplasmic reticulum and ribosomes. Randomly packed in the cytoplasm are the numerous nuclei, mitochondria and dictyosomes.
Other inclusions in the cytoplasm are vacuoles and vesicles of various types. Some of these contain a dense spherical storage body and are called the reserve vesicles. Lipid vesicles are small. In many species the oogoniail wall is smooth and colourless (P. debaryanum and P. ultimum) but in some it may be spiny (P. acanthicum).
Structure of mature oogonium:
At the time of fertilisation the oogonial contents undergo considerable reorganization. The organelles, vacuoles and vesicles move to their destination. The mitochondria, all the nuclei except one and vacuoles migrate to the periphery whereas reserve vesicles and lipid vesicles move inwards and accumulate in the central area of the oogonium.
In fact when the fertilization tube penetrates the mature oogonium its protoplast is differentiated into two zones, outer periplasm and inner ooplasm (D).
(i) Periplasm:
It is the narrow outer zone lining the oogonial wall. It contains spongy or vacuolate protoplast. Apparently it seems to have been formed by shrinking away of oogonial protoplast from the wall. In reality it is formed by the coalescence and deterioration of vacuoles which assembled there after migration.
Much of the lamellar endoplasmic reticulum accumulates around the periphery of the central dense area forming a layer parallel to the oogonial wall. The periplasm which is outside this layer contains surplus nuclei, mitochondria and other organelles.
(ii) Ooplasm:
The large, central portion of the oogonium filled with granular electron dense cytoplasm constitutes the ooplasm. It has a single nucleus and food reserve in the lipid and reserve vesicles. The uninucleate ooplasm functions as an oosphere or egg. Some mycologists hold that the uninucleate condition of ooplasm is attained by disintegration of all but one functional nucleus.
Development of antheridium:
It develops terminally on a short male or antheridial branch arising laterally either from the oogonial stalk or a neighbouring hypha (A). In its early development the antheridial branch curves towards the oogonium. It comes in contact with the oogonial wall and flattens against it with the tip getting slightly inflated (C, D).
The hyphal contents migrate into the enlarged tip. It is eventually separated from parent hypha by a hyphal plug to function as an antheridium. The antheridium in Pythium is thus applied to the side of the oogonium and is described as paragynous.
(i) Structure of antheridium:
It is an elongated, club-shaped structure much smaller in size than the oogonium. It has a thin wall. Within the antheridial wall is the plasma membrane which in the young antheridium encloses an electron-dense cytoplasm. The cell organelles (ribosomes, endoplasmic reticulum, nuclei, mitochondria and dictyosomes) are randomly packed in the cytoplasm.
Besides, there are small vaculoes. At maturity the antheridial protoplast becomes differentiated into a central uninucleate portion which functions as the male gamete and the outer periplasm. The surplus nuclei and other organelles migrate into the periplasm where they later degenerate.
Fertilization in Pythium:
The gametangial contact, as mentioned above, takes place at an early stage of development usually by the curvature of the male hypha rarely by the female hypha. The tip of the antheridium applies itself closely to the oogonial wall and becomes flattended at the point of contact.
The intervening walls of the mature sex organs dissolve at the point of contact (E). The flattened tip of the antheridium puts out a fine, tubular process known as the fertilisation or the conjugation tube.
The latter penetrates the oogonial wall through the pore, pierces the periplasm and dips into the ooplasm. Here it opens and emits (E) a single male nucleus together with a certain amount of cytoplasm. The cytoplasm mingles with cytoplasm of the egg. The male nucleus fuses with the female nucleus.
Post-fertilization Changes:
Immediately after fertilization the egg secretes a thin membrane or a continuous band of small vesicles (Haskins et al, 1976) around it. A thick wall is subsequently deposited at this layer.
The organelles involved in the synthesis and supply of material for this wall are reported to be the organelles and cytoplasm in the periplasmic space and the lamellar endoplasm reticulum layer which delimits the periplasm from the ooplasm.
With the formation of thick wall, all these structures disappear in the periplasm. Meanwhile the small reserve vesicles in the developing fertilized egg enlarge and coalesce to form a single, large, dense globule in the centre. It is surrounded by the rapidly enlarging and closely packed lipid globules of various sizes.
The resultant thick-walled structure gorged with reserve food material and scanty cytoplasm is the mature oospore.
Fine Structure of Oospore (Fig. 6.22 A):
The thin oogonial wall loosely surrounds the mature oospore. The periplasm having disappeared by now there is empty between the origonial wall and the oospore.
The oospore thus partially fills the oogonial cavity and is described as aplerotic. The mature oospore has a thick smooth wall. The oospore wall is two is differented into two layers, the outer exine and the inner intine. The exine is electron opaque. The intine is comparatively thicker and electron-transparent.
Ruben and Stanghelline (1978) make 3 layers namely:
(i) Outer thin electron opaque,
(ii) The middle thick transparent layer and
(iii) The thin inner layer with in the thick oospore wall is a centrally located large, dense spherical area surrounded by closely packed lipid globules of various sizes filling the space between the oospore wall and the central area.
The scanty cytoplasm forms a thin layer around the central structure and lies between the lipid vesicles. This central area of the oospore has been differently interpreted by various investigators. Merchant (1968) described it as the large, centrally located diploid nucleus in P. ultimum.
Hemmes and Bartnicki-Garcia termed it the ooplast. Mckeen (1975) termed this central area of the oospore as the reserve globule formed by the fusion of the reserve vesicles. According to him, the oospore nucleus is not prominent. It lies sandwiched between the lipid vesicles and reserve globule.
Haskins et al, (1976) reported that in P. acanthicum the reserve bodies in the vesicles expand and display a ‘finger print’ pattern and the central area in the mature oospore is not as large as depicted by McKeen (1975). It contains one, sometimes two nuclei and much endoplasmic reticulum.
The oospore is a resting structure. The nature of the thick oospore wall provides protection and the large amount of lipid globules furnishes energy needed for its long dormancy.
After fertilization, the antheridium adhering to the oogonial wall retains its characteristic shape as an empty or nearly empty shell. Drechsler (1946) reported parthenogenesis in Pythium. The unfertilized egg behaves like an oospore and is called parthenospore.
Oospore Germination (Fig. 6.23):
On the onset of conditions favourable for growth the oospore germinates. Prior to germination the oospore imbibes water and swells completely filling the oogonial cavity.
The diploid oospore nucleus undergoes repeated division. The increase in the number of nuclei is accompanied by increase in the number of mitochondria. The central globule begins to disintegrate and becomes surrounded by vacuoles indicating its digestion and absorption.
Simultaneously there is erosion and pitting of the middle layer of the oospore wall which becomes thin in the region of germ tube emergence. The emerging germ tube with its wall continuous with the inner layer of the oospore wall pushes through the middle and outer layers, at this site.
After emergence from the oospore wall the germ tube presses against the oogonial wall and finally breaks through it behaving in either of the following ways (Fig. 6.23):
(i) It grows into a small hypha. The latter infects the host and forms the starting point of the fresh mycelium.
(ii) The tip of the hypha swells (E) to form a vesicle-like sporangium (P. debaryanum). The entire oospore protoplast migrates into the latter. The terminal sporangium may either behave like a zoosporangium and produce the zoospores (F) or gets detached. The detached sporangium behaves like a conidium and germinates directly by putting out a germ tube.
(iii) In the third mode of germination (P. anandrum and P. mamillatum) the contents of the oospore divide to from biflagellate zoospores which are emptied into a vesicle. The vesicle soon vanishes and the zoospores are liberated. Each liberated zoospore germinates and infects the host in the usual manner and forms a new fungus mycelium.
According to Drechsler (1952), at high temperature (28°C) the oospore germinates directly by a germ tube. The latter develops into a new mycelium. At lower temperatures within a range of 10-17°C the germ tube swells at the tip to form a vesicle. The oospore protoplast migrates into the vesicle where differentiation of zoospores takes place.
Alteration of Generations in Pythium Debaryanum:
Prior to germination the diploid nucleus of the oospore divides a number of times. Edison (1915) suggested that the first two successive divisions are meiotic. On the basis of zygotic meiosis scheme (Fig. 6.24) the vegetative thallus (mycelium) of Pythium is considered haploid. It bears haploid gametangia (sex organs) containing gametes towards the end of the growing season.
The male and female gametes fuse to form the oospore which is the only diploid structure representing the diplophase (sporophyte) in the life cycle of Pythium.
All other structures, the zoospores, the mycelium, the gametangia and gametes represent the haplophase (gametophyte). Such a life cycle with a prolonged haploid vegetative phase (haplophase) and a single-celled diploid oospore representing the diplophase is called haplontic. It is characterized by zygotic meiosis and haploid adult (mycelium).
Till recently this scheme of zygotic meiosis and haplontic Pythium life cycle had been held widely by mycologists. Sansome working on P. debaryanum struck a discordant note. She advanced evidence indicating that Pythium has a diploid vegetative thallus (mycelium) and gametangial meiosis (Fig. 6.25).
According to her, the two successive divisions which occur in gametangia (oogonium and antheridium) are meiotic. Dennett and Stanghellini (1977) have also advanced genetic and cytological evidence for diploid life cycle and gametangial meiosis in P. aphanidermatum.
They have photographed the diplotenediakinesis figures in both the antheridia and oogonia. On the basis of gametangial scheme of meiosis Pythium has a prolonged diploid vegetative phase represented by the zoospores, mycelium and gametangia (oogonia and antheridia).The haploid phase is extremely reduced.
It is represented only by the gametes (male nucleus and oosphere). This kind of life cycle is called diplontic. It is characterised by gametangial meiosis and diploid adult (mycelium). The vegetative phase whether diploid or haploid is prolonged by the formation of sporangia which germinate directly or indirectly by the intervention of zoospores in the growing season.
Control of the Disease Caused by Pythium:
To control pre-emergence phase of ‘damping off, the seeds are treated with a suitable seed protectant such as Blitox-50 and Arasan. The chemical should be applied so as to form a protective layer external to the seed coat. Since moisture content of soil, humidity of air and over-crowding of seedlings are the prime factors in the spread of disease
Following sanitary measures are suggested to check it:
(i) Thin sowing to avoid over-crowding.
(ii) Adequate exposure to air and light of the seed beds.
(iii) Prevention of excessively moist conditions by providing nursery beds at a raised level to maintain good soil drainage.
(iv) Use of light, more porous and sterile soils.
(v) Use of well decomposed manure.
(vi) Lowering the humidity of the atmosphere.
(vii) Sterilisation of soil by dry heat or steam.
(viii) The soil treatment of nursery beds by the use of chemicals such as formaldehyde diluted in proportion of one part to 50 parts of water and Bordeaux mixture ( ½ gallon per square foot). ‘
(ix) Drenching soil with 0.2% Fytolan, 0.5% Perenox and 0.2% Flit 406 have been reported to provide effective check to control the ‘damping off disease.
