Czapek Dox Agar Medium Research Articles

Production of Citric Acid from Different Aspergillus species Obtained from Soil

The genus Aspergillus is considered highly important in the production of various types of enzymes and organic acids. Aspergillus species produce organic acids such as citric acid, itaconic acid, and malic acid, which are one of the most important alternate techniques for chemical processes. Citric acid is an essential primary agricultural product that is extensively used in the world. It acts as an intermediate in the tricarboxylic acid cycle (citric acid cycle) during the oxidation of carbohydrates to carbon dioxide. Citric acid has an important role in industries involving the production of food products, beverages, pharmaceutical and agricultural products, detergents, and cosmetics. The production of citric acid, a crucial agricultural commodity with widespread applications in various industries including food, beverages, pharmaceuticals, agriculture, detergents, and cosmetics, heavily relies on microbial fermentation. The great demand for citric acid due to its wide industrial applications and less toxicity. Citric acid can be produced using less expensive substrates that are renewable too. In this work, Aspergillus species was isolated from the soil source, collected in different localities of Chhatrapati Sambhajinagar. The total 90 different isolates are isolated among these the 20 different Aspergillus species are isolated and pure culture by using potato dextrose agar (PDA) Czapek dox agar medium and study the effect of different media on growth and sporulation of different Aspergillus species and the quantitative analysis of citric acid production was done by titration method with help of NaOH and phenolphthalein indicator. The main reason for the study increases is the large number of applications that can be found for citric acid, primarily in the food and pharmaceutical industries

Role of Copper Ions in Resistance of Modern Polymer Composite Materials to Fungal Damage

Resistance of polymer composite materials to biodamage is one of the pressing problems of our time. Incorporation of Cu2O (I) in the composition of a polymer composite based on the ED-20 epoxy resin increases its biocidal properties. Under conditions of mineral and organic contamination, the area of the samples affected by micromycetes was found to decrease with increasing concentration of dispersed particles in the composite. The affected area of the samples filled with the particles encapsulated in polylactide was 1.5 times smaller than that of the composites filled with non-encapsulated particles. Сopper oxide had a toxic effect on the Aspergillus niger strain dominant on the surface of the samples, causing a decrease in the average radial growth rate on the Czapek-Dox agar medium and in the biomass weight concentration during the growth of micromycetes in a liquid medium compared to the variant without Cu2O.

Effect of Different culture media, temperature and pH for growth of Rhizoctonia solani causing Sheath Blight Disease in Rice Mat Nursery

Over the last decade Sheath Blight of Rice as a major constant in rice mat nursery production has been identified globally and integrated disease resistance strategy for Sheath Blight of Rice has been primary focus in south east Asia particularly in the kharif and rabi rice growing areas like Bangladesh, Eastern India, Terai region of Nepal and Pakistan. Rhizoctonia solani (Kuhn.) is the causal agent of Sheath Blight Disease in Rice Mat Nursery. It is a soil inhabiting fungal pathogen which favors hot and humid temperature. Keeping in view the consequentiality of the disease due to transmute in climatic conditions, studies were conducted on the cultural and morphological diversity of the pathogen in different media, pH and temperature conditions. This research was conducted at Department of Plant Pathology in Uttar Banga Krishi Viswavidyalaya. The fungus Rhizoctonia solani gives highest mycelial growth on PDA (Potato dextrose Agar) media which is 60.78 mm and it is followed by Glucose peptone Agar media (55.33 mm) and V-8 juice Agar media (51.11 mm) whereas the lowest growth was observed in Czapek Dox Agar media (19.66 mm) followed by Oat meal Agar media (26.66 mm). In different pH, maximum mycelial growth was observed at pH 7 i.e., at neutral pH (62.33 mm) while they showed less growth in too acidic pH i.e., at 4 and at too high pH i.e., at pH 10. In different temperature, the highest mycelial growth was observed at 26⁰C (62.91 mm) to 28⁰C (62.05 mm) i.e., they prefer normal temperature for their growth.

Contamination of Armenian Red Pepper by Microfungi and Aflatoxin B1

Key words: paprika, hot red pepper, filamentous fungi, aflatoxin B1, strains, contamination The aim of this study was to investigate the contamination of ground red pepper with mycotoxigenic fungi. During 2019-2020 we have analyzed 45 samples taken from different regions of the republic. 13 species of filamentous fungi, belonging to five genera were isolated from ground pepper. Such spices as Aspergillus niger, A. flavus, A. fumigatus var. griseobrunes, A. ochraceus, Penicillium lanosum и Absidia corymbifera were most frequently isolated from red pepper samples on Czapek-Dox agar medium. A chromatographic analysis of 17 samples of ground red pepper was carried out for the presence of aflatoxin B1. As a result, Aflatoxin B1 had been detected in 9 samples. Aflatoxin concentrations in paprika and hot red pepper ranged from 10 to 50 μg/kg. The aflatoxin content in red pepper samples did not depend on its pungency. Other mycotoxins have not been identified during the present study. These findings indicate the risk possibility of human exposure to mycotoxins through the consumption of dry rep pepper.

Identification of Previously Unrecorded Bacills, Serratia, and Mucor Strains Isolated from Yogurt

(1) Background: Foodborne illnesses are recognized as a significant threat to public health and the economy in both developed and developing nations. The safety of foods containing microorganisms has consequently become a major worldwide concern. One of the most frequent causes of food deterioration in the world is microbial contamination. (2) Methods: Yogurt containers that were bought commercially in Riyadh, Saudi Arabia during their validity period contained four different species of bacteria and one type of fungus. Using nutritional agar and Czapek-Dox agar medium, the bacteria and fungi were isolated. The isolates of the fungi and bacteria were identified using a scanning microscope. The isolates were further identified and classified for molecular evolutionary analyses using the 16S rRNA and ITS sequences from the bacteria and fungi, respectively, in conjunction with the universal primers 27F, 1492R, ITS1F, and ITS4R. (3) Results: A total of 131 separate strains were identified from 12 yogurt samples based on their phenotypic characteristics. In total, 79 isolates (60.3%) consisted of Serratia marcescens, Bacillus subtilis, and Mucor circinelloides, with 52 isolates (39.7%) being Bacillus cereus. While the cells of Bacillus and Serratia are shaped like rods, the sporangia of Mucor are large, round, and black. Each strain was identified by its accession number, which were as follows: MK590996.1: B. cereus; MK591144.1: B. subtilis; MK591002.1 and MK591014.1: S. marcescens; and MK559692.1: M. circinelloides. The maximum identification was found to be between 98.64 and 100% when BLAST was used to compare the sequences to the NCBI GeneBank database. (4) Conclusions: Genus and species identification was performed using the similarity score values. Yogurt products containing high concentrations (39.7%) of Bacillus cereus isolates carry a significant risk of health hazards due to the potential for spreading pathogenic bacteria to humans.

The Mechanism of Ochratoxin Contamination of Artificially Inoculated Licorice Roots.

Ochratoxin (OT) contamination of medicinal herbs is a serious threat to human health. This study was performed to investigate the mechanism of OT contamination of licorice (Glycyrrhiza sp.) root. Licorice root samples were cut into eight parts, which were placed separately on sucrose-free Czapek Dox agar medium, inoculated with the spores of ochratoxigenic Aspergillus westerdijkiae. After incubation for 10 and 20 days, the OT contents of the samples were determined by high-performance liquid chromatography, and microtome sections prepared from the samples were analyzed by desorption electrospray ionization tandem mass spectrometry, to visualize OT localization. The same sections were further examined by light microscopy and scanning electron microscopy, to investigate the path of fungal mycelial penetration of the inner roots. OT concentrations tended to increase from the upper- to the middle-root parts. OTs were located in cut areas and areas of cork layer damage; they were not present in the undamaged cork layer, indicating that the structure of this layer prevents OT contamination of the licorice root.

Biological control of cabbage head rot (Sclerotinia sclerotiorum) by Coniothyrium minitans

BackgroundSclerotinia sclerotiorum is the causal agent of white mould, stem and head rot diseases on a wide variety, including cabbage in the field and storage. Control of this pathogen by using commercial disease management methods is extremely difficult. Therefore, this study was performed to identify the potential biological control agent.ResultsThe antagonist fungal isolates, a mycoparasite of S. sclerotiorum, were isolated and described as KET Cm1, PAL Cm 2 and TDK Cm 3. They were subjected to morphological and microscopic observation. The growth of antagonist fungal isolates on different media revealed that PDA and half-strength Czapek dox agar medium showed the maximum mycelial growth (28.67 and 28.33 mm) and pycnidial production in the PDA medium. The studies on pH and temperature found that growth and sporulation of the antagonist fungal isolates were optimum at pH 5.5 (34 mm) and 20 °C. The molecular confirmation of antagonist fungal isolates was carried out using ITS 1 and ITS 4 primers, which target 18S-28S rRNA gene fragment. The isolates were partially sequenced and showed that more than 99% identity with Coniothyrium minitans already exist in the NCBI database. The accession numbers (MW093134, MW111282 and MW136938) were obtained for all the three isolates and also confirmed using specific primers Cm sp 1F and Cm sp 1R. Among the tested isolates, the antagonistic activity of C. minitans TDK Cm 3 showed the maximum mycelial growth inhibition of 48.8% S. sclerotiorum and was used for further studies. Penetration and formation of appressoria on the hyphae of S. sclerotiorum by C. minitans isolate TDK Cm 3 were observed through the scanning electron microscope. The ethyl acetate fractions of TDK CM 3 isolate were evaluated against S. sclerotiorum by agar well diffusion assay, paper disc assay and poison food technique in vitro. Through GC–MS analysis, the effective antifungal compounds were identified.ConclusionThe present study focused on the identification and characterization of effective Coniothyrium isolates as eco-friendly, integrated disease management strategies against cabbage head rot disease.

PRODUCTION OF FUNGAL CELLULASE ENZYMES AND THEIR APPLICATIONS

Cellulose is the most common organic polymer. The enzyme cellulase may break it into smaller sugar components, such as glucose subunits, which can be utilized to make ethanol, organic acids, and other compounds. Cellulases are a set of enzymes that have applications in various industries, including food, textiles, pharmaceutical, detergent, biofuels, and paper, making them the third-largest industrial enzyme utilized worldwide with rising demand. Cellulolytic fungi were first screened by exposing them to Carboxyl Methyl Cellulose agar (CMC), a selective media for cellulolytic bacteria. Further testing can be done on Czapek-Dox agar medium supplemented with 1% CMC. The cellulase production was significantly improved by optimizing many parameters such as pH, temperature, and substrate concentration.

Pigment Produced by Glycine-Stimulated Macrophomina Phaseolina Is a (−)-Botryodiplodin Reaction Product and the Basis for an In-Culture Assay for (−)-Botryodiplodin Production

An isolate of Macrophomina phaseolina from muskmelons (Cucumis melo) was reported by Dunlap and Bruton to produce red pigment(s) in melons and in culture in the presence of added glycine, alanine, leucine, or asparagine in the medium, but not with some other amino acids and nitrogen-containing compounds. We explored the generality and mechanism of this pigment production response using pathogenic M. phaseolina isolates from soybean plants expressing symptoms of charcoal rot disease. A survey of 42 M. phaseolina isolates growing on Czapek-Dox agar medium supplemented with glycine confirmed pigment production by 71% of isolates at the optimal glycine concentration (10 g/L). Studies in this laboratory have demonstrated that some pathogenic isolates of M. phaseolina produce the mycotoxin (−)-botryodiplodin, which has been reported to react with amino acids, proteins, and other amines to produce red pigments. Time course studies showed a significant positive correlation between pigment and (−)-botryodiplodin production by selected M. phaseolina isolates with maximum production at seven to eight days. Pigments produced in agar culture medium supplemented with glycine, beta-alanine, or other amines exhibited similar UV-vis adsorption spectra as did pigments produced by (±)-botryodiplodin reacting in the same agar medium. In a separate study of 39 M. phaseolina isolates, red pigment production (OD520) on 10 g/L glycine-supplemented Czapek-Dox agar medium correlated significantly with (−)-botryodiplodin production (LC/MS analysis of culture filtrates) in parallel cultures on un-supplemented medium. These results support pigment production on glycine-supplemented agar medium as a simple and inexpensive in-culture method for detecting (−)-botryodiplodin production by M. phaseolina isolates.

Isolation and Identification of Penicillium rubens from the Local Strain in Mosul, Iraq, and Investigation of Potassium Phosphate Effect on its Growth.

Penicillium species is one of the most common microscopic filamentous fungi that have been isolated from widespread substrates. In this study, soil samples from different areas of Mosul, Iraq, were examined by morphological and molecular methods. Fungi were isolated and grown in diagnostic culture media, including Czapek Yeast Extract Agar, Malt Extract Agar, and 25%Glycerol nitrate agarat different temperatures of 15, 20, 25, and 30°C. The growth rate of the fungus was also evaluated in the culture containing K2HPO4. In the molecular method, the obtained fragment was sequenced using the Internal Transcribed Spacer region primers after replication. Morphological evaluation of both macroscopic and microscopic features revealed that strains were Penicillium rubens, and molecular methods used have confirmed the isolated strain. The results of this study showed a decrease in the diameters of the colonies at different growth temperatures when doubling the amount of K2HPO4. In addition, the findings revealed that compared to growth inhibition in the control groups, the maximum growth inhibition was 16 and 29 mm on Czapek Dox Agar (modified) medium at incubation temperatures of 20°C and 25°C, respectively. As a result, K2HPO4 has an important role in inhibiting growth; therefore, it can be used as Fungicide.

Antagonistic activity of resident biological agents against wilt and root rot pathogens of pea in different culture media

Antagonistic potential of rhizospheric fungal and bacterial isolates evaluated against wilt and root-rot pathogens of pea exhibited significant effect on radial growth inhibition of pathogens in comparison to control. Two isolates of Trichoderma spp., one isolate of Aspergillus niger, one isolate of Penecillium oxalicum and three isolates of Pseudomonas were isolated from rhizosphere of pea and were evaluated for their antagonistic potential against three soil borne plant pathogens viz., Fusarium oxysporum f. sp, pisi, Rhizoctonia solani and Pythium ultimum using dual culture technique in different culture media (potato carrot agar, czapek dox agar, rose bengal chloramphenicol and czapek malt agar). Antagonistic potential of fungal and bacterial isolates were found to be significantly influenced in different culture media. All fungal and bacterial isolates preferred potato carrot agar and czapek dox agar media and resulted in maximum reduction in growth of test pathogen on the culture media.

Geotrichum Candids Inciting Fruit Rot of Ivy Gourd in Synergistics Effect of Agrochemicals on Carbendazim

There was variation in the MIC of carbendazim among the Geotrichumcandidus incitant of fruit rot of Ivy gourd (Cucurbitaceae) fruits and isolate on (CZA) CzapekDox agar medium plates. Minimum inhibitory concentration (MIC) on CZA plates ranged from 4000 µg/ml while it was more resistant with resistant factor 2. Use of carbendazim with–other agrochemicals like Fungicides (mancozeb, copper oxychloride, chlorothalonil), Insecticides (phorate, chlorpyriphos and endosulfan), Herbicides (metribuzin, sodium salt and atrazine), Antibiotics (bacipen, ambistryn–s and penicillin), Salts (mercuric, chloride, stannous chloride and barium chloride), and Fertilizers (MOP, urea, 10:26:26) inhibited the growth of the pathogen in invitro studies.

Efficacy of different culture media on growth and characteristics of mycelium of monkey head mushroom (Hericium erinaceus)

Culture medium is very important for growth of fungus mycelium in laboratory under controlled conditions. The pure culture of Hericium erinaceus was obtained from Mushroom Technology Laboratory of Department of Plant Pathology, CCS HAu, Hisar. Initial culturing was done on potato dextrose agar (PDA) media. The disc (5mm in diameter) from actively growing culture was plated on fresh media plates of potato dextrose agar (PDA), malt extract agar (MEA), yeast extract agar (YEA), oat meal agar (OMA) and Czapek-Dox agar media (CDA). The growth of mycelial disc was recorded daily. The results indicated that PDA was the best medium for mycelium growth. The maximum growth was observed on PDA (90.0 mm) followed by MEA (73.7 mm) and CDA (73.7 mm) in 15 days. It was also observed that the mycelium growth was started after 4 days of inoculation on PDA, MEA and YEA, on the other hand the mycelial growth was started after 5 days on Czapek-Dox agar media. The mycelium fully covered the Petri-plate on 15th day of inoculation on PDA followed by MEA medium in 18 days then YEA in 19 days whereas in Czapek-Dox medium mycelium took 20 days to fully colonize. The density of mycelium on PDA and MEA was very thick and white in color whereas on CDA and YEM mycelium had very sparse growth, less thick and less whitish in nature. The morphological characteristics of mycelium of H. erinaceus mushroom were also recorded.

A 9-Year Experience of Aspergillus Infections from Isfahan, Iran.

PurposeAspergillosis is an important fungal disease affecting millions of individuals worldwide. The genus of Aspergillus consist of various complexes, causing a wide spectrum of diseases from superficial infections in immunocompetent hosts to life-threatening disseminated infections among immunocompromised patients. This study aimed to identify Aspergillus species by phenotypic (total isolates) and molecular tests (35 isolates), obtained from patients in Isfahan (the third-largest city of Iran) between 2010 and 2018, and determine the susceptibility of 35 clinical isolates to itraconazole (ITR), amphotericin-B (AMB), and voriconazole (VOR).Patients and MethodsBased on clinical signs, a total of 2385 suspected cases were included in this retrospective study from January 2010 to December 2018. Direct microscopic examination with potassium hydroxide, sabouraud dextrose agar with chloramphenicol, and czapekdox agar media was applied to identify etiologic agents. Thirty-five Aspergillus species collected from January 2016 to December 2018 were identified by PCR-sequencing of ITS1-5.8SrDNA-ITS2 region, and their susceptibility to ITR, AMB, and VOR was determined using E-test.ResultsBased on direct microscopy and positive culture, 132 out of 2385 suspected cases had Aspergillus infection (5.5%). Fifty-four patients were male, and 78 patients were female. Patients in the age groups of 41–50 and 21–30 years had the highest and lowest frequencies, respectively. Aspergillus flavus/oryzae (n=54), A. fumigatus (n=24), A. niger (n=15), and A. terreus (n=12) were the most prevalent Aspergillus species, respectively. Among 35 Aspergillus species, the MIC ranges of AMB, ITR, and VOR for A. flavus/oryzae, A. niger, and A. terreus were (0.5–4 μg/mL; 0.5–16 μg/mL; 0.25–8 μg/mL), (1 μg/mL, 1 μg/mL, 1 μg/mL), and (4–4 μg/mL, 0.5–1 μg/mL, 0.5–1 μg/mL), respectively.ConclusionAspergillus infections have a wide spectrum of clinical manifestations and often occur in immunocompromised patients. Accurate identification at the species level is essential since the emergence of cryptic species is connected to different patterns of AFST that affect patient treatment outcomes. Azole-resistant Aspergillus spp. is a global concern, and the detection of the route of resistance is pivotal to prevent and control infection.

EVALUATION OF POMEGRANATE PEEL AS A SUBSTRATE FOR CITRIC ACID PRODUCTION BY ASPERGILLUS NIGER

Objective: The present study aimed to evaluate dried pomegranate peels as a substrate for citric acid production by Aspergillus niger.
 Methods: The morphological study of Aspergillus niger was carried out by wet mount with lactophenol cotton blue and slide culture method. A preliminary qualitative screening of citric acid-producing ability of this fungal strain was also performed by using the Czapek-Dox agar medium containing Bromocresol green. Dried and finely powdered Pomegranate peel was used as a principal substrate for the production of citric acid by submerged fermentation. Classical method of citric acid recovery involved a precipitation technique using calcium hydroxide followed by filtration and subsequent treatment with sulphuric acid. The citric acid produced was also chemically detected and titrimetrically estimated by 0.1 N NaOH.
 Results: The present experiment demonstrated that pomegranate peels may serve as an inexpensive medium for the production of citric acid with a yield of 19.39 g/l by using Aspergillus niger.
 Conclusion: This study provided an alternative basis to recycle the fruit peel waste of pomegranate in order to achieve industrially feasible and environmentally sustainable bio-production of pharmaceutically significant citric acid.

Anti-fungal properties of Puroindoline A against Aspergillus glaucus

Aspergillus glaucus can grow in low moisture grain, and is one of the main fungi responsible for agricultural product losses. Puroindoline A (PINA) is a tryptophan-rich alkaline adiponectin that can effectively inhibit numerous plant bacteria and fungi. However, the mechanism of PINA against A . glaucus remains unclear. Herein, we found that recombinant PINA (rPINA) could inhibit A . glaucus mycelia growth on salt Czapek dox agar (SCDA) medium and spore germination on Czapek dox (CD) medium. Scanning electron microscopy revealed that incomplete morphological characteristics of both A . glaucus spores and mycelia occurred following rPINA treatment. Laser scanning confocal microscopy (LSCM) showed that rPINA could enter the interior of spores. Flow cytometry and propidium iodide (PI) staining illustrated membranes of spores were severely damaged, especially after treatment with 0.9 mg/mL rPINA for 12 h, and spores with intact membranes were reduced by 30.7%. Additionally, rPINA reduced the mitochondrial membrane potential (MMP) by 5,5′,6,6′-tetrachlorr-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1) staining, and caused DNA damage by 4′,6-diamidino-2-phenylindole (DAPI) staining. These results indicated that rPINA may damage cell membranes and DNA structure and reduce MMP, thereby inhibiting the growth of A . glaucus . The antifungal mechanism has been demonstrated in this study, and results show that rPINA has application potential in preventing postharvest loss in the agricultural industry.

Biotransformation of lanthanum by Aspergillus niger

Lanthanum is an important rare earth element and has many applications in modern electronics and catalyst manufacturing. However, there exist several obstacles in the recovery and cycling of this element due to a low average grade in exploitable deposits and low recovery rates by energy-intensive extraction procedures. In this work, a novel method to transform and recover La has been proposed using the geoactive properties of Aspergillus niger. La-containing crystals were formed and collected after A. niger was grown on Czapek-Dox agar medium amended with LaCl3. Energy-dispersive X-ray analysis (EDXA) showed the crystals contained C, O, and La; scanning electron microscopy revealed that the crystals were of a tabular structure with terraced surfaces. X-ray diffraction identified the mineral phase of the sample as La2(C2O4)3·10H2O. Thermogravimetric analysis transformed the oxalate crystals into La2O3 with the kinetics of thermal decomposition corresponding well with theoretical calculations. Geochemical modelling further confirmed that the crystals were lanthanum decahydrate and identified optimal conditions for their precipitation. To quantify crystal production, biomass-free fungal culture supernatants were used to precipitate La. The results showed that the precipitated lanthanum decahydrate achieved optimal yields when the concentration of La was above 15 mM and that 100% La was removed from the system at 5 mM La. Our findings provide a new aspect in the biotransformation and biorecovery of rare earth elements from solution using biomass-free fungal culture systems.

Effect of medium and incubation time on production of AA, DHA and EPA from Aspergillus oryzae by solid state fermentation

Lately, artificial surfactant is more often used than natural surfactant for petroleum industry. The main advantages of using natural surfactant is because the continuous availability. Single Cell Oil, which utillizes one-cell microorganism, can be a solution, such as Aspergillus oryzae, to produce AA, DHA & EPA. A. oryzae was cultivated on Potato Dextrose Agar (PDA), Czapek Dox Agar (CDA) and Malt Extract Agar (MEA), and then the incubation times are 2,4,5,6 and 7 days in optimal medium. Lipids were extracted using ethanol and n-hexane. The characterization of lipid was done by gas chromatography (GC) method. The most optimal medium is CDA with a lipid concetration of 0,18 g. The best incubation time on CDA medium was 5 days with 0,216 g lipid content containing 0,123 g unsaturated fatty acid, 0,0613 g PUFA and 0,0615 g MUFA. The unsaturated fatty acid composition produced on the 5th day was 29.2% oleate; 29.1% linoleate and ± 0.046% EPA.

Acidophilic α-Amylase Production from Aspergillus niger RBP7 Using Potato Peel as Substrate: A Waste to Value Added Approach

In the present study a fungi was newly isolated from municipal dumping area of Midnapore town, West Bengal, India for the production of α-amylase. The isolate was identified as Aspergillus niger RBP7 (KX100578.1) through phenotypic and 18S rDNA sequencing observation. It produced clear zone (6.2 mm) on Czapekdox agar medium fortified with starch (1%, w/v). Optimization of enzyme production was studied under solid state fermentation (SSF) using potato peel as a substrate by one-variable-at-a-time (OVAT) and followed with response surface methodology (RSM), which enhanced enzyme titer up to 1112.25 U/gds with an initial medium pH-3.0. The texture of the potato peel before and after the fermentation was studied by FTIR. After fermentation the cell free supernatant was significantly effective for hydrolysis of raw starchy food stuff (taro, yam, malanga and sweet potato) and also able to produce mono-meric and dimeric sugars-like glucose and maltose. These characteristics of the enzyme may be suitable for use to treat digestive dysfunction.

FIRST REPORT OF ASPERGILLUS MINISCLEROTIGENES AS A POSTHARVEST PATHOGEN OF SOYBEAN SEEDS FROM PAKISTAN

In August 2014, seeds of Glycine max in storage rooms at Lahore, Pakistan, were found to be colonized by an unknown fungus. For pathogen isolation, seeds were incubated on moist blotters, developing a green mass of fungal spores. Spores were transferred to Czapek Dox agar medium and incubated at 25±2°C. After 7 days of incubation colonies were velvety, green and floccose, consisting of white vegetative mycelium and acquired the diameter of 3-4 cm. Globose dark brown sclerotia were produced, smaller than A. flavus. Radiate conidial heads were observed that were mostly biseriate however uniseriate heads were also present. Conidiophores were hyaline, coarsely roughened and 0.9-1.2 mm in length. Vesicles were subglobose to globose, 25-40 μm in diameter, while metulae were 8-12 μm and phialides 5-8 μm long. Conidia were pale or olive green, 3.5-5 μm in diameter. Pathogen was identified as Aspergillus minisclerotigenes (FCBP1353) (Pildain et al., 2008) and differentiated from A. flavus and A. parvisclerotigenus by studying aflatoxin profiles (B1, B2, G1, and G2) using thin-layer chromatography. Amplification of DNA fragment of 650 bp was done using the universal primers ITS1/ITS4 (Batista et al., 2008) and total genomic DNA as a template (White et al., 1990). BLAST analysis of this strain (GenBank accession No. KJ564033) showed 99% similarity to different strains (JX292091, JF412776, KF841549, JF412775). Phylogenetic analysis of identified pathogen and closely related species of A. flavus group by the Maximum Likelihood Hood tree method and profiling of DNA bands examined in the members of Aspergillus section Flavi amplified by three different Inter Simple Sequence Repeat (ISSR) primers clearly confirmed the pathogen identity. Surface sterilized healthy seeds were soaked in spore suspension (104 spores ml-1) from a one week old pathogen culture for 30 s, dried and transferred onto moist blotting paper in Petri plates. Control seeds were treated with sterilized distilled water. After 7 days incubation at 25°C, 90% of seeds were colonized and re-isolation of the same pathogen confirmed the Koch's postulates. To our knowledge, this is the first report of A. minisclerotigenes seed rot from Pakistan.