Management of mung bean leaf spot disease caused by Phoma herbarum through Penicillium janczewskii metabolites mediated by MAPK signaling cascade.

Vigna radiata L., an imperative legume crop of Pakistan, faces hordes of damage due to fungi; infecting host tissues by the appressorium. The use of natural compounds is an innovative concern to manage mung-bean fungal diseases. The bioactive secondary metabolites of Penicillium species are well documented for their strong fungi-static ability against many pathogens. Presently, one-month-old aqueous culture filtrates of Penicillium janczewskii, P. digitatum, P. verrucosum, P. crustosum, and P. oxalicum were evaluated to check the antagonistic effect of different dilutions (0, 10, 20, … and 60%). There was a significant reduction of around 7-38%, 46-57%, 46-58%, 27-68%, and 21-51% in Phoma herbarum dry biomass production due to P. janczewskii, P. digitatum, P. verrucosum, P. crustosum, and P. oxalicum, respectively. Inhibition constants determined by a regression equation demonstrated the most significant inhibition by P. janczewskii. Finally, using real-time reverse transcription PCR (qPCR) the effect of P. Janczewskii metabolites was determined on the transcript level of StSTE12 gene involved in the development and penetration of appressorium. The expression pattern of the StSTE12 gene was determined by percent Knockdown (%KD) expression that was found to be decreased i.e. 51.47, 43.22, 40.67, 38.01, 35.97, and 33.41% for P. herbarum with an increase in metabolites concentrations viz., 10, 20, 30, 40, 50 and 60% metabolites, respectively. In silico studies were conducted to analyze the role of Ste12 a transcriptional factor in the MAPK signaling pathway. The present study concludes a strong fungicidal potential of Penicillium species against P. herbarum. Further studies to isolate the effective fungicidal constituents of Penicillium species through GCMS analysis and determination of their role in signaling pathways are requisite.

Isolation and identification of Pithomyces sacchari as a leaf spot pathogen of Helianthus annuus from Pakistan.

Sunflower (Helianthus annuus L.) is an important annual crop known for its edible oil. Sunflower is susceptible to many fungal diseases including rusts, rotting, mildews, and leaf spots that result in low crop yield. Presently, infected leaves with leaf spot disease symptoms were collected from Jallo Park, Botanical Garden; University of the Punjab, Canal road, and Johar Town, Lahore for pathogen/s isolation and identification. The identification was executed morphologically as well as genetically by nucleotide sequencing of rDNA using Internal spacer region (ITS) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers. Morphological characters demonstrated a rapidly growing colony on MEA reaching 5.0-5.5 cm in diameter without zonation. The mycelial growth was rough and cottony white from the front and light pink from the reverse side. Conidia were brown, verruculose, and ellipsoidal with three to five transverse septations and one longitudinal septum ranging from 15 to 30 ± 2 µm in the broadest part. Conidiophores were long, branched, septate, 70-80 × 3-4 µm in size. Based on morphological characteristics, the pathogen was identified as Pithomyces sacchari. In genetic characterization BLAST analysis of the rDNA-ITS region of the pathogen exhibited maximum (100%) homology with other P. sacchari GenBank strains. Similarly, 99% homology was found with partial glyceraldehyde 3-phosphate dehydrogenase (GAPDH). To confirm the pathogenicity, Koch's pathogenicity test was performed by inoculating artificial fungal suspension in pots and plate assays. The emergence of similar disease symptoms and re-isolation of the same pathogens verified Koch's pathogenicity postulates. Conclusively this study confirms the identification of this novel pathogen of sunflowers and necessitates the quick development of management tools.

Plant Defense System Activated in Chili Plants by Using Extracts from Eucalyptus citriodora.

Capsicum annuum L. is infected by Fusarium Wilt and causes significant yield losses in Pakistan. Biological control is an excellent and environment friendly way. Presently, the biocontrol assays were conducted in pot trials using methanolic leaf extract of Eucalyptus citriodora L. where spray of extract prior to infection provided better protection from pathogen with maximum disease control. Further, Native page electrophoresis was performed to find out difference in expression profile of enzyme which revealed that control and T2 (Plant sprayed with Eucalyptus extract) did not exhibit any difference in their isozyme profile signifying no extra load of biological control measure on plant for the production of defense elements until the pathogen arrived. While in case of T3 （Protective treatment） and T4 (Curative treatment) extra isozyme (PO1) was observed in T4 only, PPO1 and PPO5, and PAL 2 and PAL 3 were comprised in higher quantities in T3 and T4 over control exposing the expression of plant metabolism under pathogen attack. The study concludes that the organic extract of E. citriodora have the potential to restrain the disastrous effects of pathogenic fungi. It will lead to the different aspect of biocontrol to suppress the plant pathogenic fungi in a broad spectrum.

Benzenedicarboxylic acid upregulates O48814 and Q9FJQ8 for improved nutritional contents of tomato and low risk of fungal attack.

BACKGROUND: Tomato is an important food item and a cocktail of phytonutrients. In the current study, metabolites from a non-pathogenic fungal species Penicillium oxalicum have been exploited to obtain nutritionally augmented tomato fruits from the plants to better withstand against Alternaria alternata infection. RESULTS: Initially, bioactivity-guided assay and chromatographic analyses identified the bioactive metabolites of P. oxalicum [benzenedicarboxylic acid (BDA) and benzimidazole]. Then, ≥3 times elevated quantities of vitamins and other nutritional elements (protein, fat, fibers, and carbohydrates) were achieved by the foliar application of BDA. The maximum increase (625.81%) was recorded in riboflavin contents; however, thiamine showed the second highest enhancement (542.86%). Plant metabolites analysis revealed that jasmonic acid contents were boosted 121.53% to significantly enhance guaiacyl lignin defenses along with the reduction in coumarin contents. The protein profile analysis explored three most actively responding protein species toward BDA applications, (i) palmitoyltransferase protein Q9FLM3; (ii) serine/threonine-protein kinase O48814; and (iii) E3 ubiquitin-protein ligase Q9FJQ8. The O48814 improved plant defenses; whereas, Q9FJQ8 protein was negatively regulating cysteine-type endopeptidase activity and assisted plant to resist schedule alterations. Tomato cultivar with more active innate metabolism was found to be more responsive toward BDA. Furthermore, the bioactive compounds were enriched by using the two-step extraction method of ethyl acetate and chloroform, respectively. CONCLUSION: Penicillium oxalicum a non-pathogenic fungal species, produced BDA, induced nutritional contents in tomato and protected it against Alternaria alternata. The current study is the first report on the bioactivity of BDA and benzimidazole concerning the nutritional enhancement and plant defense improvement. © 2019 Society of Chemical Industry.

Analysis of Antagonistic Potential of Secondary Metabolites and Organic Fractions of Trichoderma Species against Alternaria Alternata.

Role of Trichoderma species is well documented as antagonists as well as plant growth enhancers. Presently, the fungicidal potential of three Trichoderma species namely, T. koningii (FCBP769) , T. viride (FCBP904) , and T. harzianum (FCBP1277) was assessed against Alternaria alternata that causes leaf necrotic spots of Syzygium cumini and broad range of other plants using 0, 15, 30, 45, 60 and 75% dilutions of filtrates. There was a significant reduction of around 40-95%, 22-86% and 52-91% in fungal biomass by T. koningii, T. viride and T. harzianum, respectively. In fractionation bioassays, Trichoderma metabolites were partitioned using organic solvents viz., n-butanol, n-hexane, chloroform and ethyl acetate. Antifungal activity at different concentrations (10, 20, 30, 40 and 50 ppm) was assessed against the pathogen. Ethyl acetate fraction of T. koningii extract displayed the most promising activity resulting in 10-90% suppression in biomass. In case of T. viride butanol fraction proved the most effective in retarding the growth of pathogen from 20 to 80%. While T. harzianum extract revealed 55-85% arrest in fungal biomass due to n-hexane fraction. Present study concludes that test Trichoderma species demonstrated a strong fungicidal activity against A. alternata. Current research offers the possibility of developing strategies for controlling pathogens with bioactive metabolites of Trichoderma.

Biochemical and Molecular Screening of Varieties of Chili Plants that are Resistant against Fusarium Wilt Infection.

Pakistan holds the position of top chilies producers. So Capsicum annuum L. production in Pakistan should be promoted by combating against diseases. The only solution is to cultivate resistant varieties. Presently six chili varieties were treated with Fusarium oxysporum Schlecht. and screened for the most resistant and the most susceptible varieties. Representative varieties were evaluated for their biochemical and transcriptional profiles to discover the bases of antifungal-resistance. Results concluded that the most resistant variety was "Dandicut" and the most susceptible was "Ghotki". Tannins, coumarins, flavonoids, phenolics, Riboflavins and saponins were observed in higher quantities in Dandicut as compared to Ghotki. Defense related enzymes i.e. polyphenol oxidase, phenyl ammonia lyase and peroxidase were found in elevated amounts in Dandicut than in Ghotki. Transcriptional results showed that defense related genes i.e. PR2a, acidic glucanase; Chitinase 3, acidic; Osmotin-like PR5 and Metallothionein 2b-like had higher expressional rates in Dandicut. Pearson's correlation coefficient revealed stronger direct interaction in signal transduction and salicylic acid pathway. Resistance of chili varieties is salicylic acid based. Results obtained from this study not only help to improve chili production in Pakistan but also facilitate variety development operations. Moreover, it also constructed a scale to evaluate innate resistance among varieties.

Tomato Plant Proteins Actively Responding to Fungal Applications and Their Role in Cell Physiology.

The pattern of protein induction in tomato plants has been investigated after the applications of pathogenic and non-pathogenic fungal species. Moreover, particular roles of the most active protein against biological applications were also determined using chromatographic techniques. Alternaria alternata and Penicillium oxalicum were applied as a pathogenic and non-pathogenic fungal species, respectively. Protein profile analysis revealed that a five protein species (i.e., protein 1, 6, 10, 12, and 13) possessed completely coupled interaction with non-pathogenic inducer application (P. oxalicum). However, three protein species (i.e., 10, 12, and 14) recorded a strong positive interaction with both fungal species. Protein 14 exhibited the maximum interaction with fungal applications, and its role in plant metabolism was studied after its identification as protein Q9M1W6. It was determined that protein Q1M1W6 was involved in guaiacyl lignin biosynthesis, and its inhibition increased the coumarin contents in tomato plants. Moreover, it was also observed that the protein Q9M1W6 takes significant part in the biosynthesis of jasmonic acid and Indole acetic acid contents, which are defense and growth factors of tomato plants. The study will help investigators to design fundamental rules of plant proteins affecting cell physiology under the influence of external fungal applications.

Hypersensitive response - A biophysical phenomenon of producers.

Hypersensitive response/reaction is a form of the cellular demise frequently linked alongside plant resistance against pathogen infection. Main transducers for this reaction are the intermediates of reactive oxygen and ion fluxes which are plausibly needed for hypersensitive response (Hpr Sen Rsp). An immediate and enormous energy production and its intra-cellular biochemical conduction are imperative for an Hpr Sen Rsp to be occurred. A number of studies proved that there are such diverse types of factors involved in triggering of Hpr Sen Rsp that morphologies of dead cells have become a vast topic of study. Hpr Sen Rsp could play a frolic role in plants as certain programmed cellular disintegrations in other organisms, to restrict pathogen growth. In fact, Hpr Sen Rsp can be involved in all types of tissues and most of the developmental stages.

Cytological and physiological basis for tomato varietal resistance against Alternaria alternata.

BACKGROUND: Since tomato is an important food component, it is imperative to enhance its yield against the activities of many devastating fungal pathogens such as Alternaria alternata. The exploitation of plant innate resistance by cultivation of resistant varieties is an effective measure in this regard. In the present study, 28 tomato varieties were tested against 32 A. alternata isolates, and representative varieties were further evaluated to determine the extent and basis of their antifungal resistance. RESULTS: A significant increase (104.7%) in polyphenols was recorded in the resistant variety Dinaar compared with the susceptible variety Red Tara. Dinaar also exhibited 100% enhancement of alkaloids and terpenoids along with a 30.7% increase in cell wall hemicellulose content. Significant differences were found in physical barriers (cellulose, lignin and pectin) of the representative varieties when stained tissue sections were subjected to colorimetric analysis. Similarly, polyphenol oxidase, peroxidase and phenylalanine ammonia lyase showed increases of 78.37, 114.67 and 125.11% respectively in the resistant variety. Higher expression of glucanase genes was evident from native gel analysis, in which not only the number of isozymes but also the quantity of individual isozymes was significantly increased. CONCLUSION: The resistant variety Dinaar had strong antifungal resistance and can therefore be recommended as suitable for cultivation in the agricultural system of Pakistan.

Kinetic study of partially purified cellulase enzyme produced by Trichoderma viride FCBP-142 and its hyperactive mutants.

Cellulases are the enzymes that cleave beta-1,4 linkages of cellulose, and carbohydrate that is main part of plants' cell walls. Presently, cellulase isolation and partial purification was executed through ammonium sulfate precipitation. The isolated protein of parental and derived mutants conferred molecular weights of 30, 45 and 55 kDa. The optimum temperature for maximal cellulase activity was 50 degrees C with Ea for substrate hydrolysis of 77.73, 83.97 and 83.14 kJ mol(-1) and temperature quotient of 1.0020, 1.0022 and 1.0022 by Trichoderma viride FCBP-142, Tv-UV-5.6 and Tv-Ch-4.3, respectively. The enzyme was stable at 50 degrees C for about 60 min but rapid denaturation occurred above 55 degrees C. The enzyme showed optimum activity at pH 4.0 and involved two types of acidic and basic limbs with pKa1 and pKa2. The pKa1 of active site presented a significant shift from 2.55 to 2.9 and 3.1 by Tv-UV-5.6 and Tv-Ch-4.3, respectively in comparison to parental strain. Likewise, pKa2 moved from 6.05 to 6.5 and 6.4. Enzyme kinetics displayed Michaelis-Menten constant Km 0.6, 0.5 and 0.28 mg mL(-1) and Vmax value of 8.33, 10 and 9.09 Units mL(-1) for parental, Tv-UV-5.6 and Tv-Ch-4.3, respectively.

Management of Parthenium hysterophorus (Asteraceae) by Withania somnifera (Solanaceae).

This study was designed to evaluate the herbicidal activity of Withania somnifera (L.) Dunal against the noxious weed parthenium (Parthenium hysterophorus L.). In a laboratory bioassay, the effect of aqueous, methanol and n-hexane shoot and root extracts of 5%, 10%, 15% and 20% w/v concentrations (on a fresh weight basis) of W. somnifera were tested against the germination and seedling growth of parthenium. In general, aqueous and methanol extracts markedly suppressed the germination, root and shoot growth of parthenium. The shoot extracts were more inhibitory than the root extracts. In a foliar spray bioassay, the aqueous and methanol shoot extracts of 10% w/v (on a dry weight basis) concentration were sprayed on 1-week and 2-week-old pot-grown parthenium seedlings. Two subsequent sprays were carried out 5 and 10 days after the first spray. The aqueous and methanol extracts significantly reduced the length and biomass of parthenium shoots. In a soil amendment bioassay, the crushed shoots of W. somnifera were incorporated in the soil at 1-5% w/w. Parthenium seeds were sown one week after the residue incorporation and plants were harvested 40 days after sowing. All the soil amendment treatments significantly reduced seed germination by 43-89%. The highest dosages of 4% and 5% significantly suppressed the root and shoot biomasses of parthenium. This study concludes that foliar spray of aqueous and methanol extracts, and soil amendment with leaf residue of W. somnifera, can control the germination and growth of parthenium, one of the world's worst weeds.

Herbicidal activity of flavonoids of mango leaves against Parthenium hysterophorus L.

This study was undertaken to investigate the herbicidal activity of mango (Mangifera indica L.) leaves against parthenium weed (Parthenium hysterophorus L.). The aqueous leaf extract at 15% concentration (on fresh weight basis) significantly reduced germination, shoot length and the shoot and root biomasses of parthenium seedlings. In a leaf residue incorporation pot trial, 2% residue incorporation treatment significantly suppressed the root and shoot biomasses of parthenium, while a 5% residue treatment completely stopped the growth of the weed. Five flavonoids, namely (-)-epicatechin-3-O-ß-glucopyranoside (1), 5-hydroxy-3-(4-hydroxyphenyl)pyrano[3,2-g]chromene-4(8H)-one (2), 6-(p-hydroxybenzyl)taxifolin-7-O-ß-D-glucoside (tricuspid) (3), quercetin-3-O-α-glucopyranosyl-(1 → 2)-ß-D-glucopyranoside (4) and (-)-epicatechin(2-(3,4-dihydroxyphenyl)-3,4-dihydro-2H chromene-3,5,7-triol (5) were isolated from mango leaves. In a laboratory bioassay, 50 ppm solution of compounds 3 and 4 caused yellowing of the parthenium seedlings. A 250 ppm solution of compound 4 also significantly reduced germination and the root and shoot lengths of parthenium seedlings. This study concludes that compound 4 exhibits herbicidal activity against parthenium weed.

Molecular characterisation of UV and chemically induced mutants of Trichoderma reesei FCBP-364.

Cellulases are a highly diverse group of enzymes whose function is crucial to the healthy functioning of the biosphere, since more than half of all biomass on the planet consists of their substrate, cellulose. Trials were conducted to study the effect of mutagenesis by ultraviolet (UV) irradiation (5-40 min) and ethyl methane sulphonate (EMS) treatment (50-300 microg mL(-1)) to obtain hyperactive cellulase enzyme producers. Putative mutants of Trichoderma reesei FCBP-364 were selected on the basis of their bigger hydrolysing zone formation and compared to the parental strains quantitatively. UV- and EMS-treated putative mutants of the test strains exhibited a 1.5-2-fold enhancement in enzymatic activity over the parental strain. The profile of genetic variability among native and mutant derivatives was scrutinised through random amplification of polymorphic DNA-polymerase chain reaction (RAPD-PCR). The scanned amplicons confirmed the modification in genetic make up which might be the cause of the stir up in the enzyme activity.

Herbicidal effects of extracts and residue incorporation of Datura metel against parthenium weed.

The present study was designed to evaluate the herbicidal activity of Datura metel against the noxious weed parthenium (Parthenium hysterophorus L.). In a laboratory bioassay, the effect of aqueous, methanol and n-hexane shoot and root extracts of 5, 10, 15 and 20% w/v (on a fresh weight basis) concentration of D. metel were tested against the germination and seedling growth of parthenium. Both aqueous and methanol extracts markedly suppressed the germination and seedling growth of parthenium. Generally, the effect of shoot extracts was more pronounced than the effect of root extracts. In foliar spray bioassay, aqueous and methanol shoot extracts of 10% w/v (on dry weight bases) concentrations were sprayed on one-week and two-week-old pot-grown parthenium seedlings. Two subsequent sprays were carried out at five day intervals each. Both the aqueous as well as the methanol extracts significantly suppressed shoot length as well as shoot and root biomass of one-week and two-week-old parthenium plants. In residue incorporation bioassay, crushed shoots of D. metel were incorporated in the soil at 1, 2, ... 5% w/w. Parthenium seeds were sown one week after residue incorporation and plants were harvested 40 days after sowing. Incorporation of 2-5% residues significantly reduced germination by 47-89%. Residues of 4 and 5% concentration significantly suppressed plant biomass by 90 and 97%, respectively. The present study concludes that root and shoots of D. metel contain herbicidal constituents for the management of parthenium weed.

Alpha-amylase production by toxigenic fungi.

This study is concerned with the screening of Alternaria alternata (Fr.) Keissler and Alternaria tenuissima (Kunze ex Pers.) Wilts strains for the biosynthesis of alpha-amylases. Nine strains of A. alternata and three strains of A. tenuissima were grown on enzyme production medium (EPM) and potato dextrose agar (PDA) using three pH levels (4.5-6.5); then the selected strains, able to produce bigger zones of starch hydrolysis on solid media, were subjected the testing of their amylolytic efficacy in liquid medium. In primary screening, the amylolytic activity of all the strains was tolerant to a wide range of initial culture pH values (4.5-6.5). Of all the cultures tested, A. alternata strains FCBP-100 and FCBP-385, and A. tenuissima strains FCBP-183 and FCBP-252 exhibited the maximum potential in terms of starch hydrolysis at pH 4.5 on EPM, and hence were selected for further studies. In secondary screening, the optimum pH of fermentation medium was adjusted to 4.5 using 0.05 M citrate buffer for the estimation of amylolytic enzyme activities. At 48 h incubation, the maximum alpha-amylase activity (31.8 units mL(-1)) was discerned by A. tenuissima strain FCBP-252.

Herbicidal activity of Withania somnifera against Phalaris minor.

Herbicidal activity of Withania somnifera (L.) Dunal. was studied against Phalaris minor Retz., one of the most problematic weeds of wheat in Pakistan. In laboratory bioassays the aqueous, methanol and n-hexane extracts of 5, 10 and 15% w/v (fresh weight basis) of the roots and shoots of W. somnifera were applied. Extracts in the different solvents exhibited markedly variable herbicidal activities against germination and seedling growth of the target weed species. The methanol extracts showed the highest toxicity. Different concentrations of methanol shoot and root extracts declined the germination of P. minor by 21-71%, its shoot length by 40-72%, its root length by 50-99% and the plant biomass by 32-83%. The aqueous extracts proved to be comparatively less toxic than the methanol extracts, where generally the highest concentration of 15% exhibited pronounced toxicity against the target weed species. There was up to 48, 51, 99 and 55% suppression of the weed's germination, shoot length, root length and plant biomass, respectively, due to the 15% aqueous root and shoot extracts. Generally, the n-hexane extracts of both roots and shoots exhibited insignificant or stimulatory effects against weed shoot length and plant biomass. In a foliar spray bioassay, aqueous and methanol shoot extracts of 10% w/v (dry weight basis) concentration were sprayed on one- and two-week old pot grown P. minor seedlings. Two subsequent sprays were carried out at five day intervals each. The aqueous extract significantly reduced the shoot and root dry biomass of one-week old P. minor plants. In a residue incorporation bioassay, crushed shoots of W. somnifera were incorporated in the soil at 1, 2, ... 5% w/w. Phalaris minor seeds were sown one week after residue incorporation and plants were harvested 45 days after sowing. The lower concentrations of 2 and 3% significantly reduced, while higher concentrations of 4 and 5% of residue incorporation completely arrested, the germination of P. minor. The present study concludes that both roots and shoots of W. somnifera contain herbicidal constituents against P. minor.

Condition stabilization for Aspergillus niger FCBP-198 and its hyperactive mutants to yield high titres of alpha-amylase.

A number of substrates were tested for the cultivation of microorganisms to produce a host of enzymes. The effect of different substrates (wheat and rice straw, sugar cane waste, wood waste), incubation temperatures (20-40 degrees C), initial pH levels (3.5-9.0), incubation periods (0-72 hours) and nitrogen sources (ammonium sulfate, urea, peptone, yeast extract, sodium nitrate) on growth and alpha-amylase activity was studied for the native and mutant strains. Maximum enzyme activity was observed at 1.5% wheat straw for Aspergillus niger FCBP-198 and An-Ch-4.7 and at 2% wheat straw for An-UV-5.6, with sodium nitrate as a principle nitrogen source. The optimum temperature for maximum enzyme activity was 30 degrees C for the parental strain, while An-UV-5.6 and An-Ch-4.7 thrived well at 32.5 degrees C. The best conditions of pH and incubation duration were 4.5 and 48 hours, respectively, for all the strains. Mass production under preoptimized growth conditions demonstrated the suitability of wheat straw for swift mycelial colonization and viability.

Mutagenesis and genetic characterisation of amylolytic Aspergillus niger.

Aspergillus niger FCBP-198 was genetically modified for its ability to reveal extra cellular alpha-amylase enzyme activity. From 76 efficient mutants isolated after ultraviolet (UV) irradiation, An-UV-5.6 was selected as the most efficient UV mutant, with 76.41 units mL(-1) of alpha-amylase activity compared to wild (34.45 units mL(-1)). In case of ethyl methane sulphonate (EMS), among 242 survivors, 74 were assayed quantitatively and An-Ch-4.7 was found to be the most competent, as it exhibited a three-fold increase in alpha-amylase activity (89.38 units mL(-1)) than the parental strain. Genetic relationships of the mutants of A. niger FCBP-198 were analysed with a randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). Results obtained from the comparison between genotypes of A. niger FCBP-198 showed differences in the sizes and numbers of amplified fragments per primer for each isolate. The dendrogram showed that genotypes An-Ch-4.7 and An-Ch-4.2 were distinctly classified into one category, while the isolates An-UV-5.6, An-UV-5.1 and A. niger FCBP-198 have the nearest genetic relationship. The five isolates from A. niger FCBP-198 genotypes shared an average of 65% bands.

Strain improvement and genetic characterization of indigenous Aspergillus flavus for amylolytic potential.

Aspergillus flavus FCBP-231, a filamentous fungus, was genetically modified for its ability to reveal extra cellular alpha-amylase activity. For strain improvement, the selected strains were subjected to UV irradiation (5-40 min exposure) and EMS treatment (50-300 microg mL(-1)) for hyper activity of an alpha-amylase enzyme. The mutants were quantitatively compared with the parental strain. UV and chemical mutagenesis brought about a dramatic enhancement in enzymatic activity. The mutant strains Af-UV-5.3 and Af-Ch-5.7 exhibited 79 and 110% more enzyme activity than the native strain A. flavus FCBP-231. This improvement in enzyme activity of the mutants suggests that they are suitable strains to be used in biotechnology. RAPD-PCR analysis revealed different patterns of amplicons of native as well as mutant derivatives, which suggested that the mutation imparted changes in the genetic make up of the mutants probably involved enzyme production control.

Mutation of Alternaria tenuissima FCBP-252 for hyper-active alpha-amylase.

Production of extracellular alpha-amylase enzyme by a filamentous fungus, Alternaria tenuissima was studied in solid-state fermentation (SSF) as well as submerged fermentation (SmF). The potential strain was successfully mutated by UV and ethyl methanesulfonate (EMS). High-level of alpha-amylase activity was obtained by the mutant At-Ch-5.6 (76.75 Units mL(-1)) after chemical treatment followed by UV mutant At-UV-2.8 (63.12 Units mL(-1)) which was significantly higher than parental A. tenuissima FCBP-252 (32 Units mL(-1)). These mutants with high levels of activity were genetically characterized using RAPD-PCR. Expression pattern of mutants exhibited that the mutants were isogenic variants of parent strain and out-performance of the mutants could be attributed to change in genetic make up. This work represented the first report of strain improvement in Alternaria for hyper activity of alpha-amylase enzyme and suggested that this fungus could be used to extract purified enzyme.