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Cyclopiazonic Acid Biosynthesis of Aspergillus flavus and Aspergillus oryzae  [PDF]
Perng-Kuang Chang,Kenneth C. Ehrlich,Isao Fujii
Toxins , 2009, DOI: 10.3390/toxins1020074
Abstract: Cyclopiazonic acid (CPA) is an indole-tetramic acid neurotoxin produced by some of the same strains of A. flavus that produce aflatoxins and by some Aspergillus oryzae strains. Despite its discovery 40 years ago, few reviews of its toxicity and biosynthesis have been reported. This review examines what is currently known about the toxicity of CPA to animals and humans, both by itself or in combination with other mycotoxins. The review also discusses CPA biosynthesis and the genetic diversity of CPA production in A. flavus/oryzae populations.
Monitoring MVOC Profiles over Time from Isolates of Aspergillus flavus Using SPME GC-MS  [PDF]
Dongdi Sun, Alicia Wood-Jones, Wenshuang Wang, Chris Vanlangenberg, David Jones, Patrice Simmons, Richard E. Baird, Todd E. Mlsna, Julie Gower
Journal of Agricultural Chemistry and Environment (JACEN) , 2014, DOI: 10.4236/jacen.2014.32007
Abstract: Fungi produce a variety of microbial volatile organic compounds (MVOCs) during primary and secondary metabolism. The fungus, Aspergillus flavus, is a human, animal and plant pathogen which produces aflatoxin, one of the most carcinogenic substances known. In this study, MVOCs were analyzed using solid phase microextraction (SPME) combined with GCMS from two genetically different A. flavus strains, an aflatoxigenic strain, NRRL 3357, and a non-aflatoxigenic strain, NRRL 21882. A PDMS/CAR SPME fiber was used over 30 days to observe variations in MVOCs over time. The relative percentage of individual chemicals in several chemical classes (alcohols, aldehydes, esters, furans, hydrocarbons, ketones, and organic acids) was shown to change considerably during the varied fungal growth stages. This changing chemical profile reduces the likelihood of finding a single chemical that can be used consistently as a biomarker for fungal strain identification. In our study, discriminant analysis techniques were successfully conducted using all identified and quantified MVOCs enabling discrimination of the two A. flavus strains over the entire 30-day period. This study underscores the potential of using SPME GCMS coupled with multivariate analysis for fungi strain identification.
Fine De Novo Sequencing of a Fungal Genome Using only SOLiD Short Read Data: Verification on Aspergillus oryzae RIB40  [PDF]
Myco Umemura, Yoshinori Koyama, Itaru Takeda, Hiroko Hagiwara, Tsutomu Ikegami, Hideaki Koike, Masayuki Machida
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0063673
Abstract: The development of next-generation sequencing (NGS) technologies has dramatically increased the throughput, speed, and efficiency of genome sequencing. The short read data generated from NGS platforms, such as SOLiD and Illumina, are quite useful for mapping analysis. However, the SOLiD read data with lengths of <60 bp have been considered to be too short for de novo genome sequencing. Here, to investigate whether de novo sequencing of fungal genomes is possible using only SOLiD short read sequence data, we performed de novo assembly of the Aspergillus oryzae RIB40 genome using only SOLiD read data of 50 bp generated from mate-paired libraries with 2.8- or 1.9-kb insert sizes. The assembled scaffolds showed an N50 value of 1.6 Mb, a 22-fold increase than those obtained using only SOLiD short read in other published reports. In addition, almost 99% of the reference genome was accurately aligned by the assembled scaffold fragments in long lengths. The sequences of secondary metabolite biosynthetic genes and clusters, whose products are of considerable interest in fungal studies due to their potential medicinal, agricultural, and cosmetic properties, were also highly reconstructed in the assembled scaffolds. Based on these findings, we concluded that de novo genome sequencing using only SOLiD short reads is feasible and practical for molecular biological study of fungi. We also investigated the effect of filtering low quality data, library insert size, and k-mer size on the assembly performance, and recommend for the assembly use of mild filtered read data where the N50 was not so degraded and the library has an insert size of ~2.0 kb, and k-mer size 33.
Improved protocols for functional analysis in the pathogenic fungus Aspergillus flavus
Zhu-Mei He, Michael S Price, Gregory R OBrian, D Ryan Georgianna, Gary A Payne
BMC Microbiology , 2007, DOI: 10.1186/1471-2180-7-104
Abstract: The efficiency of the genetic transformation system for A. flavus based on uracil auxotrophy was improved. In addition, A. flavus was shown to be sensitive to the antibiotic, phleomycin. Transformation of A. flavus with the ble gene for resistance to phleomycin resulted in stable transformants when selected on 100 μg/ml phleomycin. We also compared the phleomycin system with one based on complementation for uracil auxotrophy which was confirmed by uracil and 5-fluoroorotic acid selection and via transformation with the pyr4 gene from Neurospora crassa and pyrG gene from A. nidulans in A. flavus NRRL 3357. A transformation protocol using pyr4 as a selectable marker resulted in site specific disruption of a target gene. A rapid and convenient colony PCR method for screening genetically altered transformants was also developed in this study.We employed phleomycin resistance as a new positive selectable marker for genetic transformation of A. flavus. The experiments outlined herein constitute the first report of the use of the antibiotic phleomycin for transformation of A. flavus. Further, we demonstrated that this transformation protocol could be used for directed gene disruption in A. flavus. The significance of this is twofold. First, it allows strains to be transformed without having to generate an auxotrophic mutation, which is time consuming and may result in undesirable mutations. Second, this protocol allows for double gene knockouts when used in conjunction with existing strains with auxotrophic mutations.To further facilitate functional analysis in this strain we developed a colony PCR-based method that is a rapid and convenient method for screening genetically altered transformants. This work will be of interest to those working on molecular biology of aflatoxin metabolism in A. flavus, especially for functional analysis using gene deletion and gene expression.Aspergillus flavus is a ubiquitous fungus and a plant and animal health concern. It can colonize see
Control of Aflatoxin Production of Aspergillus flavus and Aspergillus parasiticus Using RNA Silencing Technology by Targeting aflD (nor-1) Gene  [PDF]
Ahmed M. Abdel-Hadi,Daniel P. Caley,David R. F. Carter,Naresh Magan
Toxins , 2011, DOI: 10.3390/toxins3060647
Abstract: Aspergillus ?avus and Aspergillus parasiticus are important pathogens of cotton, corn, peanuts and other oil-seed crops, producing toxins both in the field and during storage. We have designed three siRNA sequences (Nor-Ia, Nor-Ib, Nor-Ic) to target the mRNA sequence of the aflD gene to examine the potential for using RNA silencing technology to control aflatoxin production. Thus, the effect of siRNAs targeting of two key genes in the aflatoxin biosynthetic pathway, aflD (structural) and aflR (regulatory gene) and on aflatoxin B1 (AFB1), and aflatoxin G1 (AFG1) production was examined. The study showed that Nor-Ib gave a significant decrease in aflD mRNA, aflR mRNA abundance, and AFB1 production (98, 97 and 97% when compared to the controls) in A. flavus NRRL3357, respectively. Reduction in aflD and aflR mRNA abundance and AFB1 production increased with concentration of siRNA tested. There was a significant inhibition in aflD and AFB1 production by A. flavus EGP9 and AFG1 production by A. parasiticus NRRL 13005. However, there was no significant decrease in AFG1 production by A. parasiticus SSWT 2999. Changes in AFB1 production in relation to mRNA levels of aflD showed a good correlation (R = 0.88; P = 0.00001); changes in aflR mRNA level in relation to mRNA level of aflD also showed good correlation (R = 0.82; P = 0.0001). The correlations between changes in aflR and aflD gene expression suggests a strong relationship between these structural and regulatory genes, and that aflD could be used as a target gene to develop efficient means for aflatoxin control using RNA silencing technology.
PRODUCTION, PURIFICATION AND SOME PROPERTIES OF EXTRACELLULAR KERATINASE FROM FEATHERS-DEGRADATION BY ASPERGILLUS ORYZAE NRRL- 447  [PDF]
THANAA H. ALI,NADIA H. ALI,LATIFA A. MOHAMED
Journal of Applied Sciences in Environmental Sanitation , 2011,
Abstract: Extracellular keratinase was produced during submerged aerobic cultivation in a medium containing chicken feather for enzyme synthesis. The en-zyme was partially purified by acetone fractionation and DEAE-Sephadex A-25 column chromatography. A purification fold about 20.7 with a yield of 37.9% as determined with keratin as substrate of the activity in crude extracts. Specific activity of this partially purified enzyme is 2312.7 U/mg. The km and Vmax values were 7.15 mM and 300U/ml respectively. The optimal pH and temperature for keratinolytic activity was approximately 7.0 and 70°C respectively. Essential amino acids like threonine, valine, methionine, isoleucine, leucine, lysine, histidine and tyrosine as well as ammonia were produced when feathers were used as substrates. Exposures of purified keratinase in absence of substrate at 80°C, for 60 minutes caused lose about 56% of its activity. This keratinase was inhibited in a variable rates by addition of EDTA, CuSO4, ZnCl2 MnCl2 and HgCl2 at a concentration of 15mM, where as iodoacetate and 2-mercaptoethanol slightly activation at the same concentration. Strain Aspergillus oryzae, therefore, shows great promise of finding potential applications in keratin hydrolysis and keratinase production.
Time course of virus-like particles (VLPs) double-stranded rna accumulation in toxigenic and non-toxigenic strains of Aspergillus flavus
Silva, Valéria N.;Durigon, Edison Luiz;Pires, Maria de Fátima Costa;Louren?o, Alexandre;Faria, Maria Jacinta de;Corrêa, Benedito;
Brazilian Journal of Microbiology , 2001, DOI: 10.1590/S1517-83822001000100013
Abstract: two strains of aspergillus flavus, non-toxigenic nrrl 6550 and toxigenic nrrl 5940, were studied over a period of 44 days, in order to detect the presence of virus-like particles (vlps) by means of electron microscopy (em) and nucleic acids electrophoresis. only the toxigenic strain contained vlps, presenting three-segmented dsrna. an increase in vlps number was observed during the exponential phase of fungal growth, up to day 12 of culture; after this, higher levels of aflatoxin production in toxigenic nrrl 5940 mycelia occurred in parallel with decreased vlps replication.
Time course of virus-like particles (VLPs) double-stranded rna accumulation in toxigenic and non-toxigenic strains of Aspergillus flavus  [cached]
Silva Valéria N.,Durigon Edison Luiz,Pires Maria de Fátima Costa,Louren?o Alexandre
Brazilian Journal of Microbiology , 2001,
Abstract: Two strains of Aspergillus flavus, non-toxigenic NRRL 6550 and toxigenic NRRL 5940, were studied over a period of 44 days, in order to detect the presence of virus-like particles (VLPs) by means of electron microscopy (EM) and nucleic acids electrophoresis. Only the toxigenic strain contained VLPs, presenting three-segmented dsRNA. An increase in VLPs number was observed during the exponential phase of fungal growth, up to day 12 of culture; after this, higher levels of aflatoxin production in toxigenic NRRL 5940 mycelia occurred in parallel with decreased VLPs replication.
Iron nutrition and virulence in Xanthomonas oryzae pv. oryzae  [cached]
M.M. ANSARI and R. SRIDHAR
Indian Phytopathology , 2012,
Abstract: The significance of iron on the growth and virulence of Xanthomonas oryzae pv. oryzae the causal agent of bacterial leaf blight of rice was examined. Lag phase was reduced with Fe supplement in the growth medium. The lag phase was
Pathogenic variability of Xanthomonas oryzae pv. oryzae  [cached]
ARVIND KUMAR*, M.N. SRIVASTAVA and N. LAKPALE
Indian Phytopathology , 2012,
Abstract: Comparison of isolates of Xanthomonas oryzae pv. oryzae on a set of isogenic rice lines under greenhouse conditions revealed that the isolates prevalent in eastern Madhya Pradesh were more virulent than those prevalent in the Punjab, India. Comparison of eastern Madhya Pradesh isolates of X. oryzae pv. oryzae on a set of differentials with isolates prevalent in the Philippines and Japan revealed differences in the pathogenicity of these isolates.
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