2. Review of literature
2.1. The genus Penicillium
Penicillium is the most well known fungal genera of the group of micro-fungi.
Penicillium can be recognised by the production of a characteristic reproductive
structure i.e. ‘penicillius’ (little brush) (Link, 1809; Haubrich, 2003). With over 200
recognised species and a ubiquitous distribution in soil, the Penicillia are one of the
largest groups of fungi and among the most common eukaryotic life forms on earth (Pitt
et al., 2000). Penicillia are familiar as blue and green moulds that occur on citrus (P.
digitatum and P. italicum) and as the maturing agent of various cheeses and meats (e.g.
P. roqueforti var. roqueforti and P. camemberti) (Geisen et al., 2001). Alexander
Fleming made the genus famous through the discovery of the antibiotic ‘penicillin’
from a culture of P. chrysogenum (P. notatum) and observed inhibiting the growth of
the pathogenic bacterium Staphylococcus aureus (Raper and Thom, 1949).
Many Penicillium species produce antibiotic compounds and also a wide range
of other biologically-active metabolites. These compounds include toxins (mycotoxins)
which can cause disease in plants, animals and humans. In soils, Penicillium species are
ubiquitously distributed and can be isolated with relative ease in the laboratory (Gomez
et al., 2007; Kirk et al., 2008). Their successful colonisation in soils is largely
attributable to their undemanding nutritional requirements and their ability to grow over
a range of temperatures, water potentials and physicochemical conditions. The ability of
Penicillium to produce a wide arsenal of biologically-active secondary metabolites is
also likely to be associated with their ability to capture and compete for resources in soil
(Kathiresann and Manivannan, 2006). Several microorganisms have been traditionally
used to produce a variety of important substances for the pharmaceutical and food
industries. Hence, primary and secondary metabolites such as peptides, enzymes,
organic acids and antibiotics produced by filamentous fungi are used for these purposes
(Bennett, 1998; Demain, 2000). The genus penicillium is one of the main sources of
these potentially active metabolites (Wright et al., 1982).
Species of Penicillium are ubiquitous soil fungi preferring cool and moderate
climates, commonly present in organic materials. The genus is commonly known as
molds, they are among the main causes of food spoilage (Samson et al., 2004). Many
species produce highly toxic mycotoxins. Some Penicillium species affect the fruits and
bulbs of plants, including P. expansum, apples and pears; P. digitatum, citrus fruits; and
P. allii, garlic. Some species are known to be pathogenic to animals e.g.
P. corylophilum,
P. fellutanum,
P. implicatum,
P. janthinellum,
P. viridicatum,
P. waksmanii and P. marneffei (Da Costa et al., 1998; Ustianowski et al., 2008).
Several species of the genus Penicillium play a central role in the production of
cheese and various meat products. Penicillium molds are found in Blue cheese. P.
camemberti and P. roqueforti are the molds on Camembert, Brie, Roquefort, and many
other cheeses. Penicillium nalgiovense is used to improve the taste of sausages and
hams, and to prevent colonization by other moulds and bacteria. In addition to their
importance in the food industry, species of Penicillium and Aspergillus serve in the
production of a number of biotechnologically produced enzymes and other
macromolecules, such as gluconic, citric, and tartaric acids, as well as several pectinases,
lipase, amylases, cellulases, and proteases including bioremediation potential (Leitão,
2009).
The genus is the source of major antibiotics. Penicillin, a drug produced by
P. chrysogenum, was discovered by Alexander Fleming in 1929, and found to inhibit
the growth of gram positive bacteria for which the Nobel Prize in Medicine was
Chemical structure of penicillin
Griseofulvin
awarded to him in 1945 (Rifkind and Freeman, 2005). Griseofulvin is an antifungal
drug and a potential chemotherapeutic agent that was discovered from P. griseofulvum.
Some other species that produce compounds capable of inhibiting the growth of tumor
cells in vitro.
2.2. Classification of Penicillia
The name Penicillium came from Penicillus (= brush) and is based on the brush
like appearance of the fruiting structures under the microscope. Penicillium produces
brush like heads. The stalk is called the conidiophore. The conidiophores are simple or
branched and are terminated by clusters of flask shaped phialides (sterigmata). The
spores (conidia) are produced in chains from the tip of the phialides, with the largest
spore at the base of the chain.
With only one exception (P. marneffei, which is thermally dimorphic), the
members of the genus Penicillium are filamentous fungi. Penicillium spp. are
widespread and are generally found in soil decaying vegetables and air. Penicillium spp.
other than P. marneffei is commonly considered as contaminants but may cause
infections, particularly in immunocompromised traits. P. marneffei is pathogenic
particularly in patients with AIDS. Its isolation from blood is considered as a HIV
marker in endemic areas. In additional to their potential, Penicillium spp. are known to
produce mycotoxins.
The taxonomic classification of Penicillium is as follows Kingdom: Fungi
Phylum: Ascomycota
Class: Eurtiomycetes
Order: Eurotiales
Family: Trichomaceae
Genus: Penicillium
A frequently cited review on the problems of Penicillium (Onions et al. 1984)
anticipated results of a multidisciplinary study to address taxonomic difficulties (Bridge
et al., 1989; Paterson et al., 1989). Identifications are still done by morphology, with a
few recent physiological and chemical/molecular methods, despite the fact that many of
the non-physiological and chemical features are regarded as subjective. Species level
identifications are often very difficult and many errors exist in the literature in
Penicillium. Many problems remain concerning the use of (a) various schemes for
names, (b) strain variation, and (c) decisions based on minutia (Onions et al., 1984).
New varieties and species have been created only to be reclassified as members of
existing taxa (Frisvad et al., 2000). Novel techniques have revealed characters which
were previously unreported in some species (Paterson et al., 2003). 16SrDNA sequence
analyses demonstrated that the subgenus Penicillium is predominately monophyletic,
and current species may be varieties (Peterson, 2000). The difficulty in identifying
filamentous fungi is highly pronounced in Penicillium, leading to misidentifications in
the literature (Dörge et al., 2000; Russell et al., 2004).
The
generally
accepted
identification
of
Penicillia
by
micro
and
macromorphological characteristics (Pitt, 1979) often gives ambiguous results,
especially for the isolates from anthropogenically disturbed and extreme habitats
(Kozlovskii et al., 1997; Kozlovskii et al., 2003; Zhelifonova et al., 2006). The
reliability of attribution of Penicillia to a certain species is of interest due to availability
of the data on species-specific production of various biologically active substances,
including alkaloids, antibiotics, mycotoxins and allergens (Tutel’yan et al., 2007). The
achievements of modern biology create prerequisites for the new schemes taking into
account the broader range of characteristics. The currently proposed novel polyphasic
taxonomy of fungi of the subgenus Penicillium employs the profiles of secondary
metabolites along with micro- and macromorphological characteristics (Samson and
Frisvad, 2004). Chemotaxonomy is based on empirical observations of the common
physiological and biochemical characteristics in phylogenetically related organisms.
The potential and actual production of secondary metabolites is a component of the
physiological and biochemical identification (Kozlovsky et al., 2009).
2.3. Ecology and distribution of Penicillium
All species of Penicillium subgenus Penicillium are able to grow at 25ºC, so
they are not psychrophiles according to the most common definitions. Most species are,
however, capable of growing at 5ºC and some are growing faster at 15 ºC. Most of the
species in Penicillium subgenus Penicillium are associated to the foods and feeds of
terrestrial animals or in some cases the dung of these animals (Frisvad et al. 2000).
However, few Penicillium species restricted to particular habitats (Filtenborg et al.,
1996). Some of these associations are so strong that they were recognized early on
(Westerdijk 1949), like the association of P. italicum and P. digitatum to citrus fruits,
but most authors have regarded the Penicillia as ubiquitous "weed" organisms (Thom,
1930; Raper and Thom 1949; Pitt, 1979). It is true that all Penicillia can be grown on
laboratory substrate e.g. malt extract agar and oat meal agar, but this does not indicate
that they are all associated to cereals, and even P. digitatum and P. italicum can grow
well on autoclaved cereal based laboratory media and do not require special media
based on citrus peels (Pitt, 1979).
Abiotic factors also play a role, including the combination of temperature, redox
potential, pH, pressure, water activity, and atmosphere (Andersen and Frisvad, 1994),
but still none of these factors can explain the association of P. italicum to citrus fruits, P.
expansum to pomaceous fruits, P. commune to cheeses and P. aurantiogriseum to
cereals. P. italicum and P. digitatum are strongly associated to citrus fruits, but if
fungicide treatment is applied, a third species, P. ulaiense, associated to these nonclimateric fruits will dominate (Holmes et al., 1994). P. expansum is the dominating
Penicillium pathogenic to apples, but if fungicides are used, P. solitum will dominate
(Pitt et al., 1991). The third known example of the influence of fungicides is natamycin
treatment that will favour P. discolor at the expense of P. commune, the otherwise
dominating species on cheese (Lund et al., 1995; Frisvad et al., 1997).
Boysen et al. (1996) reported that members of series Roqueforti are a special
case, growing very well at low pH, often in conjunction with organic acids, and at high
CO2 content of the atmosphere. This has the consequence that P. roqueforti, P. carneum
and P. paneum are the dominating species growing on rye bread, blue cheeses and
silage, but the reason for these associations may be the co-evolution with lactic acid
bacteria, which produce all the metabolic products, lactic acid, CO2 etc., that members
of series Roqueforti are easily tolerating. The reason, P. carneum is more common on
dried lactic acid fermented meat products (salami) than P. roqueforti may be that P.
carneum produces patulin, which inhibits the growth of many bacteria as reported by
Vesely et al. (1981).
P. italicum and P. ulaiense in series Italica and P. digitatum in series Digitata
are strongly associated to citrus fruits. There is no strong evidence why they are not
associated to any other plant products, although they are occasionally reported from soil
and plant roots or stems. This may be caused by cross contamination from P. digitatum
spoiling citrus fruits (Pitt et al., 1991; Holmes et al., 1994). When these fruits are
spoiled a large number of air-borne conidia are produced. Fungi isolated directly from
citrus fruits rots are most probably one of the three species listed above, and the same
species are unlikely to thrive on any other substrate.
P. camemberti, P. caseifulvum, P. commune, P. palitans and P. atramentosum
appear to be mostly associated with cheese and other milk products (Lund et al., 1995).
P. commune and P. palitans have also been reported from dried meat products and nuts,
but are much less common on such substrates. P. atramentosum has been found on
Norwegian gamalost and on other cheeses, but is much more prevalent in alkaline soils.
P. nordicum and P. nalgiovense are salt tolerant and are mostly associated to dried or
salted meat products, such as salami and dried hams (Lund et al., 1995)
P.
chrysogenum,
P.
dipodomyis,
P.
flavigenum,
P.
nalgiovense
P.
mononematosum and P. confertum are common in dry habitats and may originally have
inhabited in desert and salty soils. They are able to grow on foods at low water activities.
P. chrysogenum has been found on dried cereals, salted meat and many other low water
activity foods, but is also common in indoor air environments together with A.
versicolor (Samson et al., 2004).
Zaleski (1927) reported that P. brevicompactum, P. bialowiezense and P. olsonii
are very common in soil from tropical rain forests and soil in green-houses in other
areas of the world. These species have been found on mouldy mushroom, tomatoes,
green coffee, in processed foods, and many other substrates (Mohanty and Panda, 1998;
Nilima et al., 2007). The two former species are also common in temperate forest soil
(Zaleski, 1927), may be because of their growth of basidiocarps.
2.4. Penicillia in soil
In the soil ecosystem, Penicillium spp. are known as ubiquitous and
opportunistic saprophytes. As such, they receive their nutrition through the
decomposition of (mostly) plant material in the soil and play an important role in
fundamental process of nutrient cycling. In addition to their important role in the
recycling of organic material in soil, Penicillia are one of the relatively few groups of
microflora capable of primary weathering of soil rock and minerals. The capacity of
Penicillium spp. to solubilise (release) minerals from inorganic materials can be mostly
credited to their ability to produce an arsenal of powerful organic acids. Penicillium
species can degrade the surfaces of many rocks, including carbonate, marble and granite
(Sterflinger, 2000), serpentine (releasing silicon and magnesium), muscovite (releasing
aluminium, potassium and silicon) (Crawford et al., 2000), and basalt (Metha et al.,
1979). They are important in the bio-solubilisation of various forms of coal (Kitamura
et al., 1993, Polman et al., 1994) and can solubilize a wide range of rock-phosphates
(Whitelaw, 2000). Mineral weathering activity by Penicillium species (and other soil
inhabitants) is an important primary step in the formation of soil and transformation of
soil structure.
Penicillium is an important cellulose decomposing fungus common in tropical
forest soils. It utilizes the simplest carbohydrates and thereby plays a pivotal role in the
initiation of cellulose decomposition in a soil ecosystem (UmaDevi and Manoharachary,
1987; Mamtaz and Mishra, 1991; Panda, 2011). Many works have already been done on
occurrence and distribution of soil fungi of forest soils, some of these have dealt with
the influence of plant community type (Mohanty et al., 1991; Mohanty and Panda,
1994a; Mohanty and Panda, 1998; Manoharchary et al., 2005; Manoharchary et al.,
2008), while others have examined the effect of soil depth (Behera et al., 1991; Behera
and Mukherji, 1985; Mohanty and Panda, 1994b) and a few have attempted to examine
the diversity of these fungi (Nilima et al., 2007). Limited knowledge is available on the
abundance and diversity of soil mycobiota from tropical virgin forest soils in North East
India.
Suhail et al. (2006) reported the distribution of Penicillium spp. from the river
Indus Bed of Pakistan. Twenty four soil samples were collected from surface and the
fungi were isolated by using soil dilution and soil plate method. Of the 73 strains of
fungi isolated, 10 species of Penicillium viz., P. caesicolum (1.81%), P. commune
(1.81%), P. chrysogenum (14.73%), P. funiculosum (28.36%), P. lilacinum (4.33%), P.
notatum (12.53%), P. sclerotiorum (2.52%), P. tardum (26.47%), P. vinaceum (5.51%)
and P. roseo-purpureum (1.89%) were identified. Greater numbers of species were
isolated on soil plate method than dilution plate method. Rafi and Rahman (2002)
isolated the indigenous P. chrysogenum series from different samples comprising of
fruits, vegetables, bread and grains. Slide culture method was adopted for the
identification of fungal isolates. Only two isolates, one from spoiled mango and other
from maize were found closely related to P. chrysogenum. Most of the cultural
characteristics of P. chrysogenum isolates were observed on Sabouraud's glucose agar
medium and Czapek yeast autolysate agar medium.
2.5. Penicillia in Agriculture
2.5.1 Agents of soil-borne plant disease
Like many saprophytic fungi, Penicillium species can be weakly parasitic to
crop plants under certain conditions. Surprisingly, however, only a few have become
parasitic to actively growing plant tissue. P. gladioli cause rot of the corms of Gladiolus
and related species (McCulloch and Thom, 1928). Although many Penicillium species
may be commonly isolated from diseased plant tissue, their infestation is usually
regarded as secondary to a principal infecting agent.
2.5.2. Penicillium spp. as biocontrol agent
Strains of a few Penicillium spp. showed biocontrol activity against
Phytophthora root rot of orange (Fang and Tsao, 1995), damping-off of chickpea and
cucumber (Kaiser and Hannan, 1984; Carisse et al., 2003), Fusarium wilt of tomato (De
Cal et al., 1995) and various root rots of pea and bean (Kommedahl and Windels, 1978;
Windels, 1981). Disease suppression by Penicillium species could occur by a variety of
mechanisms: direct pathogen inhibition (antibiotic production), competition with
pathogens for energy in the soil (saprophytic competition) or for infection sites on the
root, or by inducing resistance in the plant as reported by Dewan and Sivasithamparam
(1988). Although isolates of Penicillium exhibited biocontrol activity, the metabolites
produced by these fungi were sometimes toxic to plants. Till date, there are no
commercially-available bio-control products based on Penicillium.
2.5.3. General plant growth promoters
Whitelaw et al. (1997) reported about the solubilisation of soil phosphorus
minerals by P. radicum for plant growth promotion (PGP) effect in the field. Anstis
(2004) detected in vitro production of precursors of plant hormone by P. radicum. The
microbial production of phytohormones has been shown to stimulate root branching in
the rhizosphere (Patten and Glick, 2002) resulting in significant increases in plant
growth. Similarly, the phosphate-solubilising fungus P. bilaiae has also been found to
increase production of root hairs when inoculated onto pea (Gulden and Vessey, 2000).
By increasing the root area, these Penicillium inoculants could enable plants to explore
more of the soil and the nutrients therein.
In addition to stimulating root hair production, P. bilaiae has also been shown to
increase nodulation and nitrogen uptake of pea and lentil (Gleddie, 1993). Strains of
Penicillium can be easily formulated for co-inoculation with Rhizobium in peat-based
carriers (Rice et al., 1994), and co-inoculation of legumes and this technology is
extensively used in North America. An extensive survey of root-associated Penicillium
with phosphate-solubilising activity was recently conducted in Australia (Wakelin et al.,
2004).
2.6. Molecular characterization of Penicillium spp.
Penicillium is the most frequently occurring fungus on is soil (Lund, 1995). The
growth of Penicillium species in media is different in colony diameter and colour
together with its micro morphology. Some isolates produce secondary metabolites like
penicillin, cyclopiazonic acid, rugulovasine A&B and cyclopaldic acid etc. (Lund,
1995), i.e. morphological differences and secondary metabolite profiles might be useful
methods for typing of the species. Characterization of fungal isolates is also possible
using DNA fingerprinting. PCR-based DNA fingerprinting techniques such as randomly
amplified polymorphic DNA (RAPD) analysis and amplified fragment length
polymorphism (AFLP) represents a very informative and cost-effective approach for
assessing genetic diversity of a wide range of organisms (Savelkoul et al., 1999, Vos et
al., 1995; Williams et al., 1990; Kumar et al., 2007). The usefulness of RAPD for
typing Penicillium species has been confirmed in several studies. Boysen et al. (1996)
discovered and described P. paneum as a new species divided from the blue cheese
starter culture P. roqueforti using different typing methods including RAPD analysis.
Likewise, Dupont et al. (1999) used RAPD typing as a tool for identification of the
cheese and sausage starter cultures P. camemberti and P. nalgiovense. However, RAPD
has also been useful for discrimination between Penicillium isolates below species level.
Lund and Skouboe (1998) characterized the genetic diversity and relatedness of P.
commune and P. caseifulvum isolates and they concluded that RAPD is a very robust
and reproducible method for differentiating between isolates belonging to the same
species. Similarly, Mekha et al. (1997) demonstrated that P. marneffei isolates from
Bangkok were different from a P. marneffei isolate from China using RAPD and Geisen
et al. (2001) characterized different genotypes within P. roqueforti using RAPD profiles.
Lunda et al. (2003) studied on the distribution of P. commune isolates in cheese
dairies and mapped using secondary metabolite profiles, morphotypes, RAPD and
AFLP fingerprinting. A total of 321 P. commune isolates were characterized using
morphotypes (colony morphology and colours) and secondary metabolite profiles.
Based on production of secondary metabolites, P. commune isolates were classified into
6 groups. The genetic diversity of P. commune isolates was assessed using randomly
amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism
(AFLP).
Lund (1995) differentiated Penicillium spp. by detection of indole metabolites
using a filter paper method. The indole secondary metabolites chaetoglobosin C,
cyclopiazonic acid, isofumigaclavine A and rugulovasine A and B produced by several
Penicillium species growing on Czapek yeast autolysate agar were detected directly in
the culture using filter paper wetted with Ehrlich reagent dissolved in ethanol.
Redondo et al. (2009) characterized the Penicillium spp. by Ribosomal DNA
Sequencing and BOX, ERIC and REP-PCR analysis. The genus Penicillium is one of
the largest and widely distributed of all fungal genera described to date. In the period
from the publication of the first taxonomic study on Penicillium in 1930 to the latest
classification in 2004, 225 new Penicillium spp. have been described (Thom, 1930;
Raper and Thom, 1949; Seifert and Levesque, 2004). The results of previously
published taxonomic studies, in which only morphological characteristics were used to
identify fungal species, yielded different classification proposals because of strain
variation (Pitt, 1979; Ramirez, 1982). Strain variation in a fungal species is common
due to differences in the environmental conditions of their habitat (Meyer et al., 1991).
The taxonomic studies on Penicillium can be ambiguous even using this polyphasic
approach because not all the strains from the same species display identical
characteristics or profiles (Frisvad and Samson, 2004; Smedsgaard and Nielsen, 2005).
In order to overcome the limitations of the classical microbiological techniques,
advanced molecular methods with high resolution and accuracy are now being used for
taxonomic classification of Penicillium spp. Using specific gene sequencing of
biosynthetic genes and ribosomal and mitochondrial DNA has been used to identify
new Penicillium species (Samson et al., 2004; La Guerche et al., 2007; Peterson and
Sigler, 2002; Peterson et al., 2003; Pianzzola et al., 2004; Paterson et al., 2004;
Cruickshank and Pitt, 1987; Fierro et al., 1995; Dupont et al., 1999). Despite of some
limitations, rep- PCR fingerprinting has been used to analyze the DNA of various
fungal species, and to characterize the Penicillium and other closely related fungal
genera (Edel et al., 1995; de Arruda et al., 2003; Reynaldi et al., 2003; Berg et al.,
2005).
2.7. Characterization of Penicillium isolates based on isozyme analysis
The first antibiotic Penicillin was reported to be produced by P. chrysogenum
(Raper and Thom, 1949) belonging to the P. chrysogenum series in their AsymmetricaVelutina subsection together with P. notatum Westling, P. meleagrinum Biourge and P.
cyaneofulvum Biourge and all these species were later synonymized under P.
chrysogenum by Samson et al. (1977). Several strains of P. chrysogenum were being
used for penicillin production in laboratory scale. The species are common in dried
foods, including sausages, soils and indoor air (Gravesen, et al., 1994).
Stolk et al. (1990) suggested that a species used in fermenting sausages i.e. P.
nalgiovense Laxa, was closely related to P. chrysogenum, possibly as a domesticated
form which could produce penicillin (Andersen and Frisvad, 1994). P. chrysogenum var.
dipodomyis and P. avigenum also reported to be a producer of penicillin (Frisvad et al.,
1987).
The taxonomic relationships among these species are not known but their
common ability to produce penicillin suggests a close relationship. Genetic data
obtained from isozyme analysis can be used in the construction of a more
comprehensive taxonomy of fungi (Taylor, 1993). Isozymes have been applied to
several taxonomic problems in mycology (Micales et al., 1986; Rosendahl and Sen,
1992) and isozyme patterns have previously been used in the taxonomy of Penicillia as
zymograms of extracellular enzymes (Cruickshank and Pitt, 1987; Pitt and Cruickshank,
1990).
Genetic distances between species can be estimated from the differences of
isozymes analysis (Rogers, 1986). The aim of the present study was to clarify the
taxonomic relationship among some isolates of Penicillia from local sources by
comparing genetic distances among the species together with the basic data related to
the morphological and cultural characteristics. Moreover, the present study may further
provide an opportunity to evaluate the isolated series of P. chrysogenum for the
penicillin production potential. The main objective of the isozyme typing is to study the
diversity within the species of Penicillium distributed in certain natural habitats.
2.8. Mycotoxins produced by Penicillium spp.
The terverticillate Penicillia are well known for their mycotoxin production
(Frisvad and Filtenborg, 1983). The first terverticillate Penicillia shown to be toxigenic
were P. cyclopium and P. viridicatum. P. crustosum was reported to produce penitrem
A (Pitt, 1979b, Frisvad, 1989). Penitrem A was reported to be produced by strains of P.
crustosum (Pitt, 1979b, Frisvad, 1989). Cyclopiazonic acid, cyclopiamine and
cyclopiamide were produced by P. griseofulvum (Frisvad 1989). P. viridicatum were
reported to produce ochratoxin and also citrinin although these strains proved to be P.
verrucosum or P. nordicum (Walbeek et al., 1969; Ciegler et al., 1973; Frisvad and
Filtenborg 1983). These strains producing citrinin and ochratoxin A were all P.
verrucosum (Frisvad, 1989). P. viridicatum has also been claimed to produce viridicatin
(Cunningham and Freeman 1953) and viridicatic acid (Birkinshaw and Samant, 1960);
however, the first isolate was P. solitum and the second was P. crustosum (Frisvad,
1989).
Penicillic acid was found in P. aurantiogriseum, P. aurantiocandidum, P.
cyclopium, P. freii, P. melanoconidium, P. neoechinulatum, P. polonicum, P. tricolor
and P. viridicatum. It probably increased the nephrotoxicity of ochratoxin A as this has
been shown experimentally in pigs (Frisvad et al., 2004c). All the members of series
Viridicata can produce penicillic acid and occur in cereals together with P. verrucosum
and thus ochratoxin A and penicillic acid often co-occur. However, P. verrucosum has
never been found in warm habitats.
Several reports have indicated that pigment production in submerged culture
was affected by numerous environmental factors, particularly the nitrogen source and
medium pH (Carels and Shepherd, 1997; Chen and Johns, 1993; Hamdi et al., 1996;
Hamdi et al., 1997). The growth medium, its pH and temperature had strong influences
on the growth, sporulation and conidial discharge of the fungal species (Vogelgsang and
Shamoun, 2002). Molds are affected by all the environmental factors; (chemical and
physical). Physical and chemical factors have a pronounced effect on diagnostic
characters of fungi. Fungal growth (spore germination, vegetative growth and
sporulation) has a specific set of conditions that is optimal. Important conditions in this
set are nutrient types and concentrations, light, temperature, oxygen and water
availability (Kuhn and Ghannoum, 2003). However, the effect of environmental factors
on growth of fungi is generally less specific and restricted than the effect on secondary
metabolite production (Northolt and Bullerman, 1982).
Temperature, water activity (aW) and pH were considered to be some of the
most important factors in fungal growth and differentiation (Mcmeekin and Ross, 1996).
In the present study, optimal culture conditions for the production of red pigment by
Penicillium species were investigated in shake flask and batch fermenters. Therefore,
the objectives of this study were to provide information on the effects of culture media,
its pH and temperature on mycelial growth, conidial discharge and pigment production
of three Penicillium species collected from the natural virgin soils.
2.9. Insecticidal activity of Penicillium spp.
The characterized crude extracts of seven Penicillium isolates were bioassayed
against Spodoptora littoralis larvae for weight reduction and mortality. Five extracts
caused significant reductions in weight and four of these caused significant increase in
mortality, both at the p< 0.05 level as reported by Paterson et al. (1987). Trichorderma
and Penicillia were among the genera recommended as biological agents for
Stromatium fulvum larvae (woodborer), (Hassan et al., 1988). Two new natural products
were isolated from culture broth of P. brevicompactum. Two analogues have shown
interesting insecticidal and three others have exhibited broad-spectrum fungicidal
activities (Cantin et al., 1998). Nester (1992) reported that spores, crude extracts and the
purified compound can impair the viability of immature mosquitoes (Aedes aegypti)
indicates that P. citrinum might represent an effective mosquito control agent.
P. simplicissimum (ATCC 90288) grown on okara (the insoluble residue of
whole soybean) has produced insecticidal okaramines, convulsive penitrem A and 6bromopenitrem E against silkworm (Hayashi et al., 1997). From the culture extract of P.
simplicissimum, a potent insecticidal indole alkaloids okaramines A (1), B (2) were
isolated (Shiono et al., 1999). Organic extracts from mycelium and culture broth of 21
Penicillium isolates have been tested for insecticidal, insect anti-juvenile hormone (antiJH) and antifungal activities. Culture broth extracts were the most active, mainly against
insects; nearly 25% of them have shown high entomotoxicity (100% mortality at
100 g/cm2). A strong in vivo anti-JH activity against Oncopeltus fasciatus Dallus was
detected in the culture broth extracts from P. brevicompactum p79 and p88 isolates
(Castillo et al., 1999). Extracts from cultures of P. funiculosum have been reported to
induce 80-100% mortality of the insect Panonychus ulmi Toch (Santamarina et al,
1987). Extracts of P. oxalicum and T. rifai showed 80-100% mortality to Oncopeltus
fasciatus (Santamarina et al., 1987).
2.10. Antifungal activity from Penicillium spp.
Three new piperazine metabolites were isolated from cultures of P.
brevicompactum showed strong antifungal activity (William et al., 1990). P. oxalicum
reduced vascular wilts caused by Verticillium sp. and Fusarium oxysporum under glass
house and field conditions. P. funiculosum were more effective in the biocontrol of
azalea root rot caused by P. cinnamomithan. It also reduced sweet orange (Citrus
sinensis) root rot caused by P. citrophthor (Fang and Tsao, 1995). P. oxalicum has been
reported to be a biocontrol agent for F. oxysporum f.sp. lycopersici (De cal et al., 1997).
2.11. Pharmaceuticals produced by Penicillium spp.
Interest in extrolites from species included in Penicillium subgenus Penicillium
started early with the isolation of the antibiotic mycophenolic acid by Gosio (1889).
This compound was later shown to be a potent pharmaceutical used as an
immunosuppressing agent in organ transplantations (Bentley, 2000). Mycophenolic acid
produced by P. bialowiezense, P. brevicompactum, P. carneum, P. roqueforti. P.
notatum, P. viridicatum, P. citrinum, P. aurantiogriseum and P. waksmanii showed
distinguished antimicrobial activities against clinical pathogens. P. griseofulvum has
been reported to be an important inhibitor of bacterial growth and this ability has been
associated with griseofulvin and patulin production (Jimenez et al., 1988).
Of particular interest is penicillin, which was first discovered in a strain firstly
identified as P. rubrum by Fleming (1929) and later re-identified as P. notatum (a
synonym of P. chrysogenum) and later again isolated from a strain of P. chrysogenum.
Subsequently, it was observed that all strains examined of P. chrysogenum
produced penicillin (Andersen and Frisvad, 1994) and furthermore that the other closely
related species in series Chrysogena were also produced penicillin, i.e. P. dipodomyis, P.
nalgiovense and P. flavigenum (Frisvad et al., 1987; Banke et al., 1997). Another
penicillin producer in subgenus Penicillium was P. griseofulvum (Laich et al., 2002).
Many other extrolites was found in the terverticillate Penicillia as lead
compounds, and time will show if any of these have a future as important
pharmaceuticals. Some of the commonly known antibiotics produced by the different
groups of organisms are listed below-
A list of the microbial species that produce different antibiotics are shown belowMicroorganisms
Antibiotics
Bacillus licheniformic
Bacitracin
Cephalosorium acremonium
Cephalosporin C
P.s chrysogenum
Penicillins
Streptomyces antibiotics
Actinomycin, Oleandomycin
Streptomyces griseus
Indolmycin, Streptomycin, Candicidin
Streptomyces kanamyceticus
Kanamycin
Streptomyces fradiae
Neomycin
Streptomyces albinogen
Puromycin
Streptomyces sioyaensis
Siomycin
Streptomyces lavendulae
Streptothricin
Bacillus subtillis
Bacillin, subtillin
Streptomyces cinnamonesis
Monensin
Streptomyces veneguelae
Chloramphenicol
Streptomyces verticillatus
Mitomycin
Penicillin griseofulvin
Griseofulvin
Penicillin urticae
Patulin
Pseudomonas aureofaciens
Pyrrolnitrin
Streptomyces caelestis
Celesticetin
Streptomyces sp. X-53
Echinomycin
Streptomyces cacaoi
Polyonins L & M
Streptomyces sp. P-8648
Viridogrisein
Stretpomyces sp.
Novobiocin
Micromonospora sp.
Micromonosorin
Thermophilic actinomycetes
Thermomycin, Thermocyridin, Refcin
Streptomyces spinosus
Spinosad
Streptomyces hygnoscopicus
Rapamycin
Streptomyces pencetius
Avermectin
Streptomyces erythrea
Erythromycin
Fumiquinazolline-f (P. corylophilum) was isolated from the active chloroform
extract of Czapek fermentative medium after 144 hours of incubation. The minimal
inhibitory concentration (MIC) values for M. luteus and S. aureus were 99 g/ml and
137 g/ml, respectively. The largest number of conidia was obtained after 5 days of
incubation in oat medium and the highest level of antimicrobial activity was produced
when the fungus culture was developed in the Czapek medium (Silva et al., 2004).
2.12. Screening of secondary metabolites from Penicillium spp.
Among the fungi, Penicillium spp. had the potential for production of several
bioactive molecules. The first discovered antibiotic Penicillin was derived from P.
notatum by Alexander Flemming in 1929. Although hundreds of fungi have been found
to produce antibiotic compounds, very few of them were found in wider application e.g.
Penicillin (P. chrysogenum), Cephalosphorin (Cephalosphorium acremonium) and
Griseofulvin (P. patulum) being manufactured in large quantities.
The discovery and development of antibiotics was one of the most significant
advances in medicine in the 20th century. Nevertheless, many antimicrobial agents that
were used to treat a variety of human infectious disease are now ineffective. To ensure
that effective drugs have to be available in the future, therefore it is necessary to
improve the antimicrobial use patterns and to diverse strategies to identify new
antibiotics through previously unexplored targets (Smith and Jarvis, 1999). Based on the
earlier studies done (unpublished data), Smith and Jarvis (1999) isolated few
Penicillium spp. from soil, their both intracellular and extra cellular metabolites were
assayed against stored product insects, fungi and bacteria (Wright et al., 1992; Cantin et
al., 1998). The screening of Penicillium organic extracts is a starting point for research
programs focused to the isolation, identification and synthesis of useful bioactive
compounds. The genus Penicillium is selected because they have been described as one
of sources of these potentially active metabolites (Elias et al., 2006; Zhelifonova et al.,
2010).
A large number of fungal extracts and extracellular products have been found to
have antimicrobial activity, mainly the filamentous fungus Penicillum spp. Since the
discovery of penicillin, the micromycetes have been famous as producers of antibiotics
and other secondary metabolites with biological activity. Kurobane et al. (1981)
reported that P. brefeldianum produced fulvic acid which possessed antiviral, antifungal,
antioxidant and antibiotic activities. Maskey et al. (2003) isolated two active substances
8-O-methylaverufin and 1,8-Odimethylaverantin as new antifungal agents from P.
chrysogenum. Nam et al. (2000) found that compounds 8-Omethylsclererotiorinamine
isolated from P. multicolour showed antimicrobial activity. Funiculosin isolated from P.
funiculosum possessed antibiotic activity (Ando et al., 1969), as well as substance SQ
30,957, a new antibiotic produced by P. funiculosum (Singh et al., 1986). Some
compounds have been isolated from P. ochrochloron as penitremi A, B, C, D, E and F,
brom-penitrem A and F, dehydro-penitrem D, but without antibacterial analyses
(Nielsen and Smedsgaard, 2003). Stamatis et al. (2005) reported inhibitory effect of
some micromycetes on the growth of Helicobacter pylori (clinical isolates). Of the 22
micromycetes tested, the most active were also found to be P. ochrochloron and P.
funiculosum with MIC comparable to those of clarithromycin, while the active
metabolite (−) 2,3,4-trihydroxybutanamide showed MIC ranging from 0.97 to 3.9
µg/mL.
P. chrysogenum (formerly, P. notatum) is an important industrial organism due
to its ability to produce several
-lactam antibiotics, particularly penicillins. The
discovery of P. notatum by Alexander Fleming and the production of the revolutionary
drug, penicillin, was perhaps the most important finding in the history of therapeutic
medicine. Two naturally occurring and commercially available penicillins are Benzyl
penicillin (Penicillin G) and Phenoxy-methyl penicillin (Penicillin V). The R-group
substituent of the penicillin nucleus can be substituted to give the molecule different
antibacterial properties. The antibacterial effect of -lactam antibiotics is effectively
nullified by different types of bacteria which produce
-lactamase, an enzyme that
breaks the -lactam ring. Clinical isolates of extended-spectrum -lactamase (ESBL)producing bacteria have been reported in different regions of the world. Efforts at
improving penicillin yields have centred on growth optimization, development of
available strains of P. chrysogenum by classical mutagenesis procedures, and the search
for better strains of the organism (Kozlovsky et al., 1997 & 2003).
2.13. Penicillin from Penicillium spp.
Penicillin discovery by Fleming’s legendary observations on the inhibition of
bacterial growth by P. notatum (Fleming, 1929) was one of the greatest advances in
medicine (Bycroft and Shute, 1987). This is due to the importance that this antibiotic
assumed during the Second World War and even today as it continues to be a source of
many ß-lactam semisynthetic derivatives of clinical use (Demain and Elander, 1999;
Elander, 2003; Lein, 1986). Large-scale penicillin production in 1941 was solved by the
isolation of new strains, selection of improved derivatives and optimization of the
fermentation technique (Backus and StauVer, 1955; Raper et al., 1944). P.
chrysogenum is still the species of choice for industrial penicillin production. Many
mutant strains became available from the improvement programs that raised penicillin
productivity more than 1000-fold (Elander, 2003; Lein, 1986). This was the beginning
of the chapter of the antibiotic production of a fungal species to the present day’s
molecular improvement of penicillin production.
Dayalan et al. (2011) explained about the comparative study on production and
purification of penicillin by P. chrysogenum isolated from soil and citrus samples. Up to
1986 not much was known about the penicillin biosynthesic machinery. Carr et al.
(1986) described the cloning of the pcbC gene encoding isopenicillin N synthase from P.
chrysogenum, catalyzing the crucial oxidation step that forms the ß-lactam nucleus.
Most of the available literature related to penicillin and Penicillium was focused on the
complex biochemistry of the enzymes involved and the genetics behind the increased
productivity of industrial P. chrysogenum strains. It was reported that this increased
productivity was for a large part caused by a unique amplification of a set of clustered
genes (Barredo et al., 1989; Fierro et al., 1995). This was confirmed by transforming
wild type strains with isolated and/or combined genes of the penicillin biosynthetic
cluster which resulted in derivatives with increased penicillin productivity (Veenstra et
al., 1991; Fernandez-Canon and Penalva, 1995; Kennedy and Turner, 1996). Fierro et al.
(1995) showed that the amplified region in all industrial strains was much larger than
only the three penicillin biosynthetic genes.
Wenzler et al. (2003) studied the in vitro activity of penicillin G compared with
penicillin and other antibiotics against common organisms causing ear, nose and throat
(ENT) infections. Muñiz et al. (2007) showed that other then P. chrysogenum, P.
nalgiovense and P. olsonii were found to be positive to penicillin production in an agar
assay and further examination for antibiotic production in liquid culture with complex
media designed for penicillin production, confirmed their ability for penicillin
biosynthesis. Penicillin production by P. olsonii was reported for the first time by
Muñiz et al. (2007).
Today’s industrial penicillin production strains are derived, not from Fleming’s
original isolate of P. notatum but from a higher-producing isolate of P. chrysogenum
that was discovered on a rotting cantaloupe at the Northern Regional Research
Laboratories, Illinois (Rowlands, 1991). This was the parental strain in a program for
titre improvement carried out at the University of Wisconsin, where strains such as Q176 were produced. Subsequently, most of the significant strain and process
improvement was carried out by the in-house laboratories of pharmaceutical companies
which are unpublished for commercial secrecy (Rowlands, 1991).
Kurobane et al. (1981) reported that P. brefeldianum produced fulvic acid which
possessed antiviral, antifungal, antioxidant and antibiotic activities. Maskey et al.
(2003)
isolated
two
active
substances
i.e.
8-O-methylaverufin
and
1,8-
Odimethylaverantin as new antifungal agents from P. chrysogenum. Nam et al. (2000)
found that compounds 8-Omethylsclererotiorinamine isolated from P. multicolour
showed antimicrobial activity. Funiculosin isolated from P. funiculosum possessed
antibiotic activity (Ando et al., 1969), as well as substance SQ 30,957, a new antibiotic
produced by P. funiculosum (Singh et al., 1986). Some compounds were isolated from
P. ochrochloron as penitremi A, B, C, D, E and F, brom-penitrem A and F, dehydropenitrem D, but without antibacterial analyses (Nielsen and Smedsgaard, 2003).
Stamatis et al. (2005) reported inhibitory effect of some micromycetes on the growth of
Helicobacter pylori (clinical isolates). Of the 22 micromycetes tested, the most active
were also found to be P. ochrochloron and P. funiculosum with MIC comparable to
those of clarithromycin, while the active metabolite (−)2,3,4-trihydroxybutanamide
showed MIC ranging from 0.97 to 3.9 µg/mL.
Penicillin biosynthesis is regulated by many factors such as carbon, nitrogen and
phosphate content of the medium (Feng et al., 1994; Stevenson et al., 2004).
Fermentation development in the laboratory needs the determination of carbon, nitrogen,
inorganics, and if necessary, complex nutrients supporting growth, and then
modifications of the medium to support product biosynthesis. Several studies revealed
that mineral nutrients, including microelements, could stimulate the secondary
metabolism in fungi (Lovkova et al., 1995). Mineral salts (NaCl, KCl, MgSO4, Feso4,
MnCl2, ZnSo4, CaCl2 and CuSo4) are usually used in enzymatic production (Marsafy et
al., 1977; Henning et al., 2005; Virupakshi et al., 2005), and they studied the effect of
media composition on the penicillin production, and reported that the penicillin activity
was not affected, due to the addition of carbonate (0-1%). They also reported that the
omission of cupric, magnesium, manganese, zinc sulphates and acetic acid did not affect
the penicillin activity, while the omission of ammonium nitrate and potassium
dihydrogen phosphate decreased the penicillin activity in the medium.
The uptake of sulphate, the first step in the pathway, has been studied by using
mycelium and isolated plasma membrane vesicles from P. chrysogenum (Hillenga et al.,
1996). Effect of different salts on Xylanase production in solid-state fermentation of
Penicillium sp. has been studied by Assamoi et al. (2008). It was assumed that potential
isolates of P. chrysogenum may exist in the soil as natural source and penicillin G may
be found higher in the quality and quantity than existing isolates of P. chrysogenum.
The coastal soils of India, especially Tamilnadu (Kumari et al., 2005; Kathiresan and
Manivannan, 2006), potentially have enormous biodiversity of Penicillium sp. However,
they have not been extensively explored to identify the commercially viable species of
P. chrysogenum strains and to evaluate their ability to form penicillin (Jayashree et al.,
1999; Patrick, 2005; Ostergaard et al., 2000). Therefore, there is need of a systematic
screening program to isolate the potential antibiotic producing strains e.g. P.
chrysogenum from different soil samples and to determine the effect of different
minerals through quantitative assays for its optimal productivity.
Form the above literature, it was clear that there is no report on the study of the
genus Penicillium and its diversity from the Indo Burma Biodiversity hot spot region of
North east India. Here, it is aimed to identify the fungal communities together with
Penicillium and to screen the antimicrobial activity of the Penicillium isolates. The
metabolites found potential in screening tests were bio-assayed against clinical bacterial
pathogens for their pharmaceutical applications.