Rhizopus oryzae

The Rhizopus oryzae species complex is a group of zygomycete fungi that are common,
cosmopolitan saprotrophs. Some strains are used beneficially for production of Asian fermented
foods but they can also act as opportunistic human pathogens.

Sample:

 Soil
 Dung
 Rotting vegetation

Culture characteristics:

When grown on agar media, all R. oryzae complex strains have similar growth patterns and growth
rates. Actively growing colonies fill a 90 mm Petri dish in 2 to 3 days at room temperature (23°C).
After 3 to 4 days, most isolates begin to produce sporangiospores, although some strains sporulate
more slowly and may require 10 to 14 days.

Morphological characteristics:
Importance:

 Rhizopus oryzae is parasitic fungi that penetrate citrus fruit tissue through micro-wounds
and bruises. High temperatures and high relative humidity favor the growth of the
organisms. The most favorable temperature is 25–40°C.
 Rhizopus oryzae can produce high levels of lactate from glucose, which is used as a food
additive or as a precursor for the production of degradable plastics
 The fungus also is able to produce L(+)-lactic acid from recycled office paper, suggesting
an excellent way for bioconversion of used paper
 Enzymes derived from Rhizopus oryzae, break down cheese curd and acid casein
 R. oryzae are used in the production of tempeh.
 Rhizopus oryzae, categorized as potentially pathogenic because of their ability to grow at
37 °C
  Bongkrek food poisoning was common in Indonesia. This illness is associated with the
consumption of bongkrek, which are flat white cakes made from pressed coconut and
fermented with the fungus Rhizopus oryzae.

 Mucormycosis is an infection caused by fungi belonging to the order Mucorales. Rhizopus

oryzae is the most common organism isolated from patients with mucormycosis and is
responsible for ∼70% of all cases of mucormycosis.
Identification of fungi:
 The fungal colonies were sub-cultured on Potato Dextrose Agar (PDA). The isolates were
identified based on their morphological and microscopic features. Two drops of cotton-
blue-in-lactophenol were placed on clean glass slide and small piece of mycelium free of
medium was removed with sterile inoculating needle and transferred on to the stain. The
mycelium was teased (picked) out with the needles and covered with clean cover slip
carefully avoiding air bubbles and observed under the microscope for vegetative and
reproductive structures.
 Filamentous fungi should be identified by morphological methods. The identification
should be made by observing at first the colony morphology, color and appearance, then
observe by microscope the asexual and/or sexual reproductive structures (conidia, spores,
hyphae, etc.). Molecular techniques may be used to confirm or support the morphological
identification, but they are too expensive and in many cases are not accurate.
Aspergillus versicolor

Sources:

 soil
 hay
 cotton
 dairy products

Cultural Characteristics:

The colonies of versicolor ususally display a wide range of colors including white tan to
yellow,green and brown.
Morphology:

Importance:

 It produces a pigment asperversin that is uses as a antifungal agent.

 It causes aspergillosis, onchomycosis and certain allergic reactions.
 A.versicolor is very effective at removing lead ions, adsorbing 45 mg of lead per gram of
dry fungal biomass.
 Aspergillus versicolor is also useful in the industrial production and purification of
xylanase, which is often used to degrade xylan in waste products from hardwood
manufacturing and agricultural activities.

Aspergillus terreus

Source:
  Soil
 Dust
 Compost

Cultural Characteristics:

The colonies of A.terreus has a

 valvety white texture

 Cinnamon-buff-brown colour on the surrounding.
 White to brown on the reverse side.

Morphology:

 Conidial heads
 Conidiophores
 Conidia

Importance:

 It is able to produce potent lipases and cellulase and secrete itaconic acid (IA).It makes the
fungus attractive for white biotechnology.
 it produces varity of natural products most notably the medically important HMG-CoA
reductase inhibitor lovastatin.
 For decades it has been used as mean to control pathogenic fungi from destroying plants.
 A range of metabolites has been extracted from Aspergillus terreus among them are
Territrems, citreoveridin, and butyrolactones.
 It also has been shown to disturb the male sexual reproductive cycle in plant model
organism Arabidopsis thaliana. It does this by producing secondary metabolite aspterric
acid that inhibit the production of pollens.
 Causes mycotic abortion in cattle.
 Causes sinusitis in dogs.
 Also cause opportunistic, superficial and sytemic type infections in humans.

Mucor
Source:

 Soil

Microscopy:

 Aseptate hyphae.
 Sporangia filled with round spores.
 Difference from rhizopus is that, rhizopus has rhizoid and stolon while mucor has no
rhizoid.

Colonies:

 White colonies on culture media

 Colonies become black when mature due to black spores.

Primary Structure:

 White colonies spotted at side of SDA plate.

Secondary Culture:

 White conies on whole plate, by giving 72 hours more incubation colonies turn to brown
and black due to spores.

Clinical significance:
  Mucor are unable to infect human and animals due to their inability to grow in warm
environment close to 37C.
 Sometime cause opportunistic and necrotizing infection known as zygomycosis.

Benefits:

 Oil producing fungus

 Large amount of chitosan rich biomass
 Autolysate can replace yeast extract in microbial cultivation
 Molecular analysis for confirmation:
 DNA Barcoding:
 The purpose of a PCR (Polymerase Chain Reaction) is to make a large number of copies
of a gene or other part of the genome. This is necessary to yield enough starting template
from the extracted DNA for further sequence analysis. During this practical we will
amplify the fungal ITS2 region to analyse the fungal communities
 . The Internal Transcribed Spacer (ITS) region of nuclear ribosomal DNA (rDNA) is now
perhaps the most widely sequenced DNA region in fungi, and has been chosen as the bar
code for fungal species identification. However it is not optimal for all fungal groups.


 The ITS region, separating 18S, 28S and 5.8S coding regions of the rDNA gene, is present
in high copy numbers allowing easy amplification of the region from total DNA. It has
typically been most useful for molecular systematics at the species level because of its high
rate of polymorphism between species. The ITS region is separated into ITS1 and ITS2,
both immediately flanking the 5.8S gene sequence, with the former upstream and the latter
 downstream of that sequence. ITS primer map. Fungal specific primer fITS7 (forward) and
universal primer ITS4 (reverse) will be used.
 There are three major steps in a PCR, which are repeated for 20 to 40 cycles. This is done
on an automated thermal cycler, which can heat and cool the tubes with the reaction mixture
and DNA template in a very short time. Because both DNA strands are copied during PCR,
there is an exponential increase of the number of copies of the gene.
 1. Denaturation at 94°C: During the denaturation, the double strand melts apart to single
stranded DNA, and all enzymatic reactions stop.
 2. Annealing at 57°C: The primers form temporary ionic bonds with the single stranded
template. The more stable bonds last a little bit longer (primers with a good fit) and on that
piece of double stranded DNA (template and primer), the polymerase can attach and starts
copying the template. Once there are a few bases built in, the ionic bond is so strong
between the template and the primer, that it does not break anymore.
 3. Extension at 72°C: This is the ideal working temperature for the polymerase. The
primers, where a few bases are already built in, already have a stronger ionic attraction to
the template than the forces breaking these attractions. Primers that are on positions with
no exact match, get loose again due to the higher temperature. The bases that are
complementary to the template, are coupled to the primer on the 3' side. The polymerase
adds dNTP's from 5' to 3', reading the template from the 3' to the 5' side; bases are added
complementary to the template.