TABLE OF CONTENTS
MATERIALS AND METHODS
Aspergillus flavus isolates
Differentiation of the isolates (aflatoxin study)
Morphological characterization of A. flavus isolates
Physiological characterization of A. flavus isolates
Biochemical characterization of A. flavus isolates
Aspergillus flavus is the most widely known species of the genus Aspergillus which is known as a species in 1809 and first reported as a plant pathogen in 1920 (Leslie, 2008). Like other Aspergillus species, this fungus has a worldwide distribution due to its numerous conidia production, which easily disperses by air movements and possibly by insects. Aspergillus flavus is mainly a saprophyte in the soil, where it plays a major role as a nutrient recycler, supported by plant and animal debris and contaminates a wide variety of agricultural products in the field, storage areas, processing plants, and during distribution. The ability of A. flavus to survive in unfavorable conditions allows it to easily out-compete other organisms for substrates in the soil or plant. It grows better with water activity (aw) ranges from 0.86 - 0.96 with an optimum temperature of 37 oC, but able to grow in the range of 12 - 48 oC. This optimum temperature contributes its pathogenicity in human being. (Hedayati et al., 2007; Ruiqian et al., 2004)
Aspergillus flavus belongs to Subgenus Circumdati sections Flavi and the taxonomy of this species is as follows:
Domain: Eukaryote Kingdom: Fungi Phylum: Ascomycota Class: Eurotiomycetes Order: Eurotiales
Family: Trichocomaceaae Genus: Aspergillus
Species: Aspergillus flavus Link.
In general, A. flavus colony appear as a velvety, yellow to green or brown mold with colorless or sandy beige reverse. Old colony appears as dark green. The shape is smooth and some have radial wrinkles. Conidiophores are heavy walled, uncolored, coarsely roughened, usually less than 1 mm (400-800 μm) in length and are often rough just beneath the globose vesicles. Vesicles are elongate when young, later becoming subglobose or globose, varying from
10 to 65 mm in diameter. Phialides are uniseriate (single layered) or biseriate (two layered). The primary branches are up to 10 mm in length, and the secondary up to 5 mm in length (Hedayati et al., 2007 & Ruiqian et al., 2004).
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Fig: 1. Characteristics of Aspergillus conidiophores (Klich, 2007)
Many fungal cultures produce toxic secondary metabolite called mycotoxin. They are broadly classified as aflatoxins, trichothecenes, fumonisins, zearalenone, ochratoxin A and ergot alkaloids, which pose the greatest potential risk to plant, human, and animal health. Some mycotoxins that are produced by A. flavus include -nitropropionic acid, aflatoxin B1, aflatoxin B2, aflatoxin B2a, aflatoxin G1, aflatoxin G2, aflatoxin M, aflatrem, aspertoxin, cyclopiazonic acid, gliotoxin, sterigmatocystin, and versicolorin A.
Another species such as A. parasiticus, A. nomius, A. pseudotamarii, A. bombycis, and
A. toxicarius also produces aflatoxin, difuranocoumarin derivative produced by a polyketide pathway. Four major types of aflatoxins have been characterized, B1, B2, G1, and G2. Aflatoxin B and aflatoxin G resemble the blue and green fluorescent observed under ultraviolet light, while the subscript designates relative chromatographic mobility. Aflatoxin M is mainly found in dairy product, and hence the M initial is taken from milk. Apart from the above types, aflatoxin B1 is the most pathogenic compound. (Hedayati et al., 2007; Klich, 2007).
Most A. flavus produces aflatoxin B1 and B2. Aflatoxin is harmful in plant, human and animal health. Some commodities that have been found contaminated are peanuts, cotton, corn, cereals, dried fruits, oilseed, wheat, rice, cottonseed, copra, nuts, coffee bean, dry bean, soybean, sorghum, barley, various foods, milk, eggs, cheese, and figs. In corn, A. flavus causes an ear rot.
In peanuts, it causes a rot in mature peanuts and a seedling disease known as yellow mould of seedlings or aflaroot. The symptoms include necrotic lesions, chlorosis above - ground parts and lack of development of secondary roots (aflaroot). In cotton, A. flavus affects cotton quality by causing boll rot. Furthermore, aflatoxin B1 has been shown to inhibit seed germination of some other crop seeds, including wheat, corn, mustard, mung and gram (Klich, 2007).
In animals, birds, fish, and mammals (young pigs, pregnant sows, dog, calf, mature cattle, sheep, cat, monkey) have been reported infected by aflatoxin. The pathological effects are hepatotoxicity (liver damage), bile duct hyperplasia, hemorrhage (intestinal tract and kidneys), and liver tumors. In human, aflatoxin causes chronic cavitary pulmonary aspergillosis (CCPA) and aspergilloma (clump of fungus in body cavity, i.e. lung), allergic bronchopulmonary aspergillosis (ABPA) and allergens, keratitis and endophthalmitis, cutaneous infection, wound infection, endocarditis and pericarditis, central nervous system (CNS) infection, rhinosinusitis, allegic fungal sinusitis (AFS) and sinus aspergilloma, osteoarticular infection, and urinary tract infection. (Hedayati et al., 2007).
Characterization study of Aspergillus flavus on its morphological, physiological, and biochemical aspect is important to identify this pathogenic fungi for the management of various human, animal, and plant diseases as well for surveillance, and other epidemiological study (Kiba et al., 2007).
Aside from the above studies, the study in enzymes produce by this fungus is important because they play a major role in host-pathogen relationship. Numerous cell wall degrading enzymes can be secreted by pathogens to breach and use the plant cell wall as nutrient sources that eventually lead to develop inedible, undesirable quality, and soft rot spoilage. Pectinases are the first enzymes secreted by fungal pathogens when they attack plant cell walls. These enzymes weaken the plant cell wall and expose other polymers to degradation by hemicelluloses, cellulose, amylase, lipase, and protease (Hindi et al., 2011). Fungi also produce catalase that reacts with and neutralize hydrogen peroxide produced by plant as one of the first defense response against infecting pathogens (Agrios, 2005).
In this study, 20 A. flavus isolates were first differentiated based on their toxic nature then they were characterized based on their morphological physiological and biochemical parameters to know is there any variation among the toxic and nontoxic isolates with respect to above said parameters.
MATERIALS AND METHODS
Aspergillus flavus isolates.
Twenty A. flavus isolates were collected from culture collection of Department of studies in Biotechnology, University of Mysore, Mysore and they were originally isolated from groundnut seeds that have been collected from different agroclimatic regions of India. The collected isolates were maintained on PDA (potato dextrose agar) slants at 4 oC for further use. Differentiation of the isolates (aflatoxin study).
All the isolates were differentiated into toxigenic and non toxigenic by their ability to produce aflatoxins using HPTLC analysis. For HPTLC analysis, A. flavus isolates were grown on PDB (50 ml) for 4 days at 28±2° C. After incubation period, broth was separated from mycelia by filtration. Aflatoxins in the broth were extracted twice with equal volume of chloroform and evaporated to dryness. The residue was redissolved in 50 μl chloroform and applied onto HPTLC plate. Twenty μl of this chloroform extract was spotted on the base line of HPTLC plate (20 x 20 cm). Spotted plates were developed in Toluene: Ethyl acetate: Formic acid (10:8:2) solvent system for approximately 20 min so that the solvent front moves about 15 cm. Plates were dried and observed under long wavelength UV (365 nm) light fitted in a black cabinet (Singh et al., 1999). Plates were observed for blue and green fluorescence at Rf-value 0.5 and 0.45 respectively. HPTLC analysis showed the presence of aflatoxin B1, B2, G1 and G2 in aflatoxigenic strains.
Morphological characterization of A. flavus isolates.
Four different culture media, PDA, CZ, YESA and AFPA were used for the morphological characterization of A. flavus isolates. Each medium was sterilized at 121 °C for 20 min and poured into pre sterilized petri dishes. Each of the isolate was inoculated on one plate of each media at the centre of the petri dish. After inoculation, all plates were incubated for seven days at 25±2 oC and observed for the macro morphological characters like colony color, colony reverse, diameter, growth, texture and margin of the colony (Batista et al., 2008).
Physiological characterization of A. flavus isolates.
Physiological characterization for all the isolates was done by growing them on PDA medium with different incubation temperatures (4, 15, 30 and 40 °C) for 7 days and observed for the growth and sporulation of the isolate.
Biochemical characterization of A. flavus isolates.
For biochemical characterization, A. flavus isolates were inoculated into five different solid media for amylase, protease, lipase, pectinase and cellulase activity. Plates were kept at 25 - 2 oC for 3-5 days and growth was observed.
Amylolytic activity. The ability to degrade starch was used as the criterion for determination of ability to produce amylases. The medium used contained NaNO3, 1 g ; K2HPO4, 1 g ; MgSO4.7H2O, 1 g ; FeSO4, 0.04 g ; soluble starch 20 g ; agar 25 g, and distilled water 1 litre. After 3-5 days of incubation, 5-8 pieces of iodine crystals were dropped onto the plates lid. A clear zone around the colony in an otherwise blue medium indicated amylolytic activity. The diameter of clear zone and diameter of fungal growth were measured.
Proteolytic activity. The medium used contained NaNO3, 2 g ; K2HPO4, 1 g ; MgSO4.7H2O, 0.5 g ; KCl, 0.5 g ; FeSO4.7H2O, 0.01 g ; sucrose 30 g ; skim milk powder 10 g ; agar 20 g, and distilled water 1 litre. After incubation, complete degradation of protein in skim milk was observed as a clearing in the somewhat opaque agar around colonies. The diameter of clear zone and diameter of fungal growth were measured.
Lipolytic activity. The medium used contained peptone, 10 g ; NaCl, 5 g ; CaCl2.2H2O, 0.1 g ; agar 20 g, Tween 20, 10 ml, and distilled water 1 litre. The formation of lipolytic enymes was seen as either a visible precipitate due to the formation of the calcium salt of the lauric acid liberated by the enzyme, or as a clearing such as precipitate around a colony due to complete degradation of the salt of the fatty acid. Sorbitan monolaurate (Tween 20) was used as a source of fatty acid.
Pectolytic activity. The medium used contained yeast extract, 2 g ; pectin, 10 g ; K2HPO4, 1.5 g ; MgSO4.7H2O, 1.5 g ; agar, 25 g, and distilled water 1 litre. Following incubation, plates were flooded with a 1% aqueous solution of hexadecyltrimethylammonium bromide (cetrimide). This reagent precipitates intact pectin in the medium and, thus, clear zones around a colony in an otherwise opaque medium indicated degradation of the pectin.
- Quote paper
- Lisa Nathalie (Author), 2011, A study on Aspergillus flavus, Munich, GRIN Verlag, https://www.grin.com/document/177288