CATHOLIC UNIVERSITY OF RWANDA

FACULTY OF SCIENCE AND TECHNOLOGY

DEPARTMENT OF BIOMEDICAL LABORATORY SCIENCE

 Discuss life cycle of:

 Zygomycetes
 Deuteromycetes
 Ascomycetes
 Basidiomycetes
 Describe
 Slide Culture Technique
 The Hair Perforation Test

 Mycotoxins And Their Related Effects

Prepared by:

MUSHIMIYIMANA Alice ICUR17AK04885

Lecturer: Mr NZUMVIRIMANA Charles

Save on 12 January 2020

ZYGOMYCETES

Zygomycetes have asexual and asexual life cycles. In the sexual life cycle, plus and minus
mating types conjugate to form a zygosporangium.When the zygospore germinates, it undergoes
meiosis and produces haploid spores, which will, in turn, grow into a new organism.

Zygomycota: The Conjugated Fungi

The zygomycetes are a relatively small group of fungi belonging to the Phylum Zygomycota.
They include the familiar bread mold, Rhizopus stolonifer, which rapidly propagates on the
surfaces of breads, fruits, and vegetables. Most species are saprobes, living off decaying organic
material; a few are parasites, particularly of insects. Zygomycetes play a considerable
commercial role. The metabolic products of other species of Rhizopus are intermediates in the
synthesis of semi-synthetic steroid hormones.

Zygomycetes have a thallus of coenocytic hyphae in which the nuclei are haploid when the
organism is in the vegetative stage. The fungi usually reproduce asexually by producing
sporangiospores
 Zygomycetes have asexual and asexual life cycles. In the sexual life cycle, plus and minus
mating types conjugate to form a zygosporangium.

The black tips of bread mold are the swollen sporangia packed with black spores (Figure 2).
When spores land on a suitable substrate, they germinate and produce a new mycelium. Sexual
reproduction starts when conditions become unfavorable. Two opposing mating strains (type +
and type –) must be in close proximity for gametangia from the hyphae to be produced and fuse,
leading to karyogamy. The developing diploid zygospores have thick coats that protect them
from desiccation and other hazards. They may remain dormant until environmental conditions
are favorable. When zygospore germinates, it undergoes meiosis and produces haploid spores,
which will, in turn, grow into a new organism. This form of sexual reproduction in fungi is called
conjugation (although it differs markedly from conjugation in bacteria and protists), giving rise
to the name “conjugated fungi.”

Advantages of slide culture:

It is a rapid method of preparing fungal colonies for examination and identification. Permits

fungi to be studied virtually in situ with as little disturbance as possible. Fungi are identified
mostly by close examination of its morphology and the characteristics it possess.

DEUTEROMYCETES 

The division Deuteromycota is also called the Fungi Imperfecti or Imperfect Fungi referring to
our "imperfect" knowledge of their complete life cycles.

The Deuteromycota are characterized by production of septate mycelium and/or yeasts, and a
sexual life cycle that is either unknown or absent. Asexual reproduction is by means
of conidia (sing.=conidium) or may be lacking

A conidium may be defined as an asexual spore that is not produced in a sporangium. Where
sexual reproduction has been determined for species in this taxon, the sexual stage is usually
referrable to the Ascomycota or Basidiomycota. Ideally, once the sexual stage has been
determined, that species should be reclassified and placed in the appropriate subdivision.

However, this did not prove to be practical since many species are known best by their asexual
stage. Thus, a compromise was reached and both the asexual and sexual stage are recognized.
As previously discussed in the Ascomycota, when both sexual and asexual stages are known to
occur in a life cycle, they are referred to as telomorph and anamorph, respectively. There are a
number of different classification schemes for this group of fungi. However, keep in mind that
since we are not working with sexual stages here that the classification schemes used to classify
the Deuteromycota is artificial and is not intended to show relationship between the taxa. We
will recognize a single class: Deuteromycetes and four orders:

Order: Moniliales

Parasexual Cycle

There are many species of Deuteromycota in which a sexual stage is not known. Of these, there
are, undoubtedly, species in which sexual reproduction occurs only in a restricted set of
environmental conditions so that the occurrence of the sexual stage is infrequent. However, it is
also apparent that some species have lost the ability to reproduce, sexually. Yet, many of the
Deuteromycota are highly successful in their environment. Since sexual reproduction is the
means by which genetic diversity is maintained in eukaryotic organisms, and diversity is the the
key to survival in species, how would a species that has apparently lost the ability to reproduce,
sexually, survive? A possible mechanism that provides an answer to this question is
the parasexual cycle. This is a process in which plasmogamy, karyogamy and haploidization
takes place, but not in any particular place in the thallus nor at any specific period during its
lifecycle.

Parasexuality was first discovered by Pontecorvo and Roper (1952) in Aspergillus nidulans.
During the parasexual cycle, the following events take place:

 Formation of heterokaryotic mycelium (Fig. 1).

 Occasional karyogamy between two nuclei to form diploid nuclei (Fig.2).

 Mitosis of 2N and 1N nuclei.

 Mitotic crossing over during mitosis of some diploid nuclei (Fig. 3).

 Haploidization (not meiosis) of some diploid nuclei (Fig. 4).

 Sorting out of new haploid strains.

 Figure 1. Heterokaryon formation refers to the condition
by which genetically different nuclei are associated in a
common cytoplasm. The most common way in which this
can occur is by anastomosis (fusion) of genetically
different hyphae (see Fig. 1a on left).  Another means by
which genetically different nuclei may enter a common
protoplasm is by mutation of one or several nuclei. We will
refer to the former in this description of parasexuality.

Following initial fusion of hyphal cells, to form a

genetically different cell, mitotic division perpetuates the
cell and mycelium that is made up of genetically, different
nuclei is formed.

Figure 2. Karyogamy and mitotic division of diploid

nuclei: Following heterokaryon formation, fusion of some
haploid nuclei that are genetically the same will fuse as
well as those that are genetically different. The latter will
result in heterozygous diploid nuclei. It is estimated that
there is one heterozygous diploid nucleus will occur per
one million haploid nuclei (Pontecorvo, 1958).

Figure 3. Mitotic Crossing Over: Figs. 3a-b. During

prophase of mitosis, mitotic crossing over can occur
between chromatids of homolous chromosomes and may
produce a unique genetic recombinant.   Fig 3c.
Recombinant chromosomes separate, during anaphase, and
give rise to nuclei that are genetically different from
existing nuclei in protoplasm. This is also a rare event,
occurring in diploid nuclei, once, in 500 mitoses.
 Figure 4. Haploidization (not meiosis) of diploid nuclei.
During mitosis, errors are common. Diploid nuclei often
form one nucleus with three copies of one chromosome

(2N+1) and the other with one copy of one chromosome (2N-1). In the latter nucleus,
the continual, sequential loss of chromosomes with two copies can occur to
eventeually give rise to a haploid nucleus. When haploidization occurs in heterozygous
diploids, the resulting haploid will result in a new genetic combination.
While the parasexual cycle appears to be a viable mechanism by which genetic recombination
occurs, many mycologist believe that it does not play a role in maintaining genetic diversity in
fungi that have lost their ability to reproduce, sexually. Instead, this has been looked upon as a
laboratory phenomenon and that heterokaryon formation, in nature, is not a common event.
Thus, the parasexual cycle must also be a rare event.

ASCOMYCETES

The ascomycetes are of particular use to humans as sources of medicinally important

compounds, such as antibiotics, for fermenting bread, alcoholic beverages and
cheese. Penicillium species on cheeses and those producing antibiotics for treating
bacterial infectious diseases are examples of ascomycetes.

Many ascomycetes are pathogens, both of animals, including humans, and of plants. Examples of
ascomycetes that can cause infections in humans include Candida albicans, Aspergillus niger and
several tens of species that cause skin infections. The many plant-pathogenic ascomycetes
include apple scab, rice blast, the ergot fungi, black knot, and the powdery mildews. Several
species of ascomycetes are biological model organisms in laboratory research. Most
famously, Neurospora crassa, several species of yeasts, and Aspergillus species are used in
many genetics and cell biology studies.

Asexual Cycle

This cycle starts with a mycelium , which is the fungi in the vegetative form that contains
branched filaments called hyphae. There are specialized hyphae, called con idiophores, that
branch off from the mycelia and are capable of producing spores.
In the next step of the cycle, the conidiophores will release their spores,
called conidia or mitospores. These spores, like the fungi in every step of the asexual life cycle,
are haploid. The conidia will undergo the process of mitosis. This is the process of cell
reproduction that creates two cells that are genetically identical to the parent cell and each other.

When the spores complete mitosis, they will remain dormant to wait for ideal environmental
conditions. Once conditions are favorable, the spores will germinate to produce a new mycelium
and the cycle starts over again.

Cycle Sexual

Let's start the sexual reproductive cycle from the point of the mycelium as well. With sexual
reproduction, there will be at least one female and one male mycelium. The male mycelium has
an antheridium and the female has an ascogonium as their sex organs. When the ascogonium and
antheridium come together, they undergo plasmogamy, which is the fusion of the cytoplasm of
ascogonium and antheridium. The interesting thing about plasmogamy is that fusion of the nuclei
does not take place at this point.

In the next step of the life cycle, the fused antheridium and ascogonium are now called
an ascocarp. Sac-like cells called asci begin to grow within the ascocarp. As the asci are
forming, ascospores, which are haploid spores, from the female and male come together in
each ascus. Ascus is just the singular form of asci, so an ascus is one of those sac-like cells, the
distinguishing feature that gives Ascomycota the nickname sac fungi. Within the asci, the
ascospores undergo karyogamy, which is the fusion of their nuclei to form a diploid zygote.

The diploid zygotes will go through the process of meiosis, which is cell division that creates
haploid daughter cells that are not identical to each other. At this point, the ascospores are
haploid again. The cells now go through mitosis to create more ascospores.
BASIDIOMYCETES 

The lifecycle of basidiomycetes includes alternation of generations. Spores are generally

produced through sexual reproduction, rather than asexual reproduction. The club-shaped
basidium carries spores called basidiospores. In the basidium, nuclei of two different mating
strains fuse (karyogamy), giving rise to a diploid zygote that then undergoes meiosis. The
haploid nuclei migrate into basidiospores, which germinate and generate monokaryotic hyphae.
The mycelium that results is called a primary mycelium. Mycelia of different mating strains can
combine and produce a secondary mycelium that contains haploid nuclei of two different mating
strains. This is the dikaryotic stage of the basidiomyces lifecyle and and it is the dominant stage.
Eventually, the secondary mycelium generates a basidiocarp, which is a fruiting body that
protrudes from the ground—this is what we think of as a mushroom. The basidiocarp bears the
developing basidia on the gills under its cap.
II.1.SLIDE CULTURE TECHNIQUE

It is a rapid method of preparing fungal colonies for examination and identification.Fungi are
identified mostly by close examination of its morphology and the characteristics it possess.
In slide cultures, we are growing the fungi directly on the slide on a thin film of agar

Principle

Fungi are inoculated in small blocks of nutrition deficient agar medium (like cornmeal agar
or potato dextrose agar), covered with a coverslip and incubated. After incubation, the coverslip
is removed from the agar block and placed on another slide to which a dye, such as lactophenol
cotton blue, may be added and observed for microscopic structures.
 Materials Required:

Culture:

7-10 day old fungal culture

Media:

Sabouraud agar

Preparation of Sabouraud agar (pH-5.6)

Sabouraud agar supplemented with aureomycin*

Peptone-10g/liter
Dextrose-40g/liter
Agar-15g/liter
*Aseptically add aureomycin ,10µg per ml, to the sterile, molten and cooled medium

Equipments:

 Sterile Petri dish

 Filter paper (9cm diameter)

 U-shaped glass rod

 Microscope slides and coverslips (Sterile)

 Sabouraud’s plate with mixed culture of fungi

 Sterile Sabouraud’s agar plate

 Lactophenol cotton blue stain

 Glass capillary tube

 Scalpel

 Inoculating needle

 Sterile distilled water

 95% ethanol

 Forceps
 Procedure:

A)    Slide Culture Preparation

 Aseptically, with a pair of forceps, place a sheet of sterile filter paper in a Petri dish.

Place a sterile U-shaped glass rod on the filter paper. (Rod can be sterilized by flaming, if held
by forceps.)

 Pour enough sterile water  (about 4 ml) on  filter paper to completely moisten it.

 With forceps, place a sterile slide on the U-shaped rod

 Gently flame a scalpel to sterilize, and cut a 5 mm square  block  of  the medium  from  the 
plate  of Sabouraud’s agar or Emmons’ medium.

 Pick up the block of agar by inserting the scalpel and carefully transfer this block aseptically
to the centre of the slide.

 Inoculate four sides  of the agar square with spores or mycelial fragments of the fungus to be
examined. Be sure to flame and cool the loop prior to picking up spores.

 Aseptically, place a sterile cover glass on the upper surface of the agar cube.

 Place the cover on the Petri dish and incubate at room temperature for 48 hours.

 After  48  hours,  examine  the  slide  under  low power. If growth has occurred there will be
growth of hyphae  and production of  spores.  If  growth  is  inadequate  and spores are not
evident, allow the mold to grow for another  24–48  hours  before  making  the  stained slides.

B)    Application of Stain

 Place a drop of lactophenol cotton blue stain on a clean microscope slide.

 Remove the cover glass from the slide culture and discard the block of agar.

 Add a drop of 95% ethanol to the hyphae on the cover glass. As soon as most of the
alcohol has evaporated  place  the  cover  glass,  mold  side down,  on  the  drop  of  lactophenol 
cotton  blue stain on the slide.  Examine the slide under microscope

Advantages of slide culture:

 It is a  rapid method of preparing fungal colonies for examination and identification.

 Permits fungi to be studied virtually in situ with as little disturbance as possible

 Fungi are identified mostly by close examination of its morphology and the characteristics it
possess. In slide cultures, we are growing the fungi directly on the slide on a thin film of agar. By
doing this, there is no need to remove a portion of the fungus from a culture plate and transfer it
to the slide. So there is  less chance for the features that are key to identification, notably the
spore-bearing structures, to be damaged.

II. 2.THE HAIR PERFORATION TEST

The hair perforation test, also known as an in vitro hair perforation test, is a laboratory test
used to help distinguish the isolates of dermatophytes, such as Trichophyton mentagrophytes and
its variants.

Dermatophytes are fungal organisms that require keratin for growth. These fungi
can cause superficial infections of the hair, skin, and nails. Dermatophytes are spread by direct
contact from other people, animals, soil, and from fomites.

Procedure

 Place hair in 20ml ofdistilled water in vial.

 Add two to three drops of 10%sterile yeast extract is added to the petri dish with hair
shafts, these hair shafts are inoculated with test fungus cultured on SDA.

 The culture is incubated at 250 C for up to 1 month during which the hairs are removed

 examine microscopically at weekly intervals by lactophenol cotton blue for the

demonstration of perforation of hair.

 Isolates of T. mentagrophytes characterized by a wedge-shaped perforation of the hair

whereas those of T. rubrum do not.

III.MYCOTOXINS AND THEIR RELATED EFFECTS

Mycotoxins  are toxic compounds that are naturally produced by certain types of moulds (fungi)
Mould growth can occur either before harvest or after harvest, during storage, on/in the food
itself often under warm, damp and humid conditions. Most mycotoxins are chemically stable and
survive food processing.
  Mycotoxins are naturally occurring toxins produced by certain moulds (fungi) and can be
found in food.

 The moulds grow on a variety of different crops and foodstuffs including cereals, nuts,
spices, dried fruits, apples and coffee beans, often under warm and humid conditions.

 Mycotoxins can cause a variety of adverse health effects and pose a serious health threat
to both humans and livestock.

 The adverse health effects of mycotoxins range from acute poisoning to long-term effects
such as immune deficiency and cancer.

Common mycotoxins that are harmful to humans are

Aflatoxins

 Aflatoxins are naturally occurring mycotoxins that are produced by many species of
Aspergillus, a fungus, most notably Aspergillus flavus and Aspergillus parasiticus.

 Aflatoxins are toxic and among the most carcinogenic substances known.

 Aflatoxins are remarkably potent, often causing disease even when ingested in minute
amounts.

 Aflatoxins can cause disease throughout the body, but are most commonly known for causing
acute or chronic liver disease and liver cancer.

 Aflatoxins  they are associated with many cancers such as liver and kidney

Ergot alkaloids

 Consuming ergot alkaloids leads to diseases characterized by nausea, vomiting, giddiness

and somnolence.

 In extreme cases it may lead to a gangrene of extremities

Fumonisin mucotoxycosis

 May lead to abdominal pain and diarrhoea.

 Chronic exposure is shown to be carcinogenic

  It also has the potential of inhibiting protein synthesis and in higher concentration to inhibit
DNA synthesis

Zearalenone mycotoxins

 The major effects of Zearalenone are on the reproduction in females, where it affects
reproductive organs and their function, eventually leading to a medical condition called
hyperestrogenism

Patulin

The acute symptoms in animals include liver, spleen and kidney damage and toxicity to the
immune system. For humans, nausea, gastrointestinal disturbances and vomiting have been
reported

Patulin is considered to be genotoxic however a carcinogenic potential has not been

demonstrated yet.

Ochratoxin A

 Is produced by several species of Aspergillus and Penicillium

The most sensitive and notable effect is kidney damage, but the toxin may also have effects on
fetal development and on the immune system
REFERENCES

Caddell, Jeremy R (2002). "Differentiating the dermatophytes". CLINICAL PRACTICE:

MICROBIOLOGY. Clinical Laboratory Science.

https://courses.lumenlearning.com/wm-biology2/chapter/zygomycota/

https://study.com/academy/lesson/ascomycota-life-cycle-classification.html

https://vlab.amrita.edu/?sub=3&brch=76&sim=693&cnt=2

Prakash, P.Yegneswaran.; Bhargava, K., 2016: A modified micro chamber agar spot slide
culture technique for microscopic examination of filamentous fungi.