Environmental and developmental factors influencing aflatoxin production by <Emphasis Type="Italic">Aspergillus flavus</Emphasis> and <Emphasis Type="Italic">Aspergillus parasiticus</Emphasis>

Aflatoxins are carcinogenic mycotoxins formed by a number of fungi in the genus Aspergillus. The major fungi responsible for aflatoxin formation in crop seeds in the field and in storage are Aspergillus flavus and A. parasiticus. This review emphasizes developmental, environmental, biological, and chemical factors that influence aflatoxin formation by A. flavus and A. parasiticus.     

Characterization of the velvet regulators in <Emphasis Type="Italic">Aspergillus flavus</Emphasis>

Fungal development and secondary metabolism are closely associated via the activities of the fungal NK-kB-type velvet regulators that are highly conserved in filamentous fungi. Here, we investigated the roles of the velvet genes in the aflatoxigenic fungus Aspergillus flavus. Distinct from other Aspergillus species, the A. flavus genome contains five velvet genes, veA, velB, velC, velD, and vosA. The deletion of velD blocks the production of aflatoxin B1, but does not affect the formation of sclerotia. Expression analyses revealed that vosA and velB mRNAs accumulated at high levels during the late phase of asexual development and in conidia. The absence of vosA or velB decreased the content of conidial trehalose and the tolerance of conidia to the thermal and UV stresses. In addition, double mutant analyses demonstrated that VosA and VelB play an inter-dependent role in trehalose biosynthesis and conidial stress tolerance. Together with the findings of previous studies, the results of the present study suggest that the velvet regulators play the conserved and vital role in sporogenesis, conidial trehalose biogenesis, stress tolerance, and aflatoxin biosynthesis in A. flavus.     

An efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation method for aflatoxin generation fungus <Emphasis Type="Italic">Aspergillus flavus</Emphasis>

Aspergillus flavus often invade many important corps and produce harmful aflatoxins both in preharvest and during storage stages. The regulation mechanism of aflatoxin biosynthesis in this fungus has not been well explored mainly due to the lack of an efficient transformation method for constructing a genome-wide gene mutant library. This challenge was resolved in this study, where a reliable and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for A. flavus NRRL 3357 was established. The results showed that removal of multinucleate conidia, to collect a homogenous sample of uninucleate conidia for use as the transformation material, is the key step in this procedure. A. tumefaciens strain AGL-1 harboring the ble gene for zeocin resistance under the control of the gpdA promoter from A. nidulans is suitable for genetic transformation of this fungus. We successfully generated A. flavus transformants with an efficiency of ～ 60 positive transformants per 10⁶ conidia using our protocol. A small-scale insertional mutant library (～ 1,000 mutants) was constructed using this method and the resulting several mutants lacked both production of conidia and aflatoxin biosynthesis capacity. Southern blotting analysis demonstrated that the majority of the transformants contained a single T-DNA insert on the genome. To the best of our knowledge, this is the first report of genetic transformation of A. flavus via ATMT and our protocol provides an effective tool for construction of genome-wide gene mutant libraries for functional analysis of important genes in A. flavus.     

Survey of Vietnamese Peanuts,Corn and Soil for the Presence of <Emphasis Type="Italic">Aspergillus flavus</Emphasis> and <Emphasis Type="Italic">Aspergillus parasiticus</Emphasis>

Aspergillus flavus and Aspergillus parasiticus cause perennial infection of agriculturally important crops in tropical and subtropical areas. Invasion of crops by these fungi may result in contamination of food and feed by potent carcinogenic aflatoxins. Consumption of aflatoxin contaminated foods is a recognised risk factor for human hepatocellular carcinoma (HCC) and may contribute to the high incidence of HCC in Southeast Asia. This study conducted a survey of Vietnamese crops (peanuts and corn) and soil for the presence of aflatoxigenic fungi and used microsatellite markers to investigate the genetic diversity of Vietnamese Aspergillus strains. From a total of 85 samples comprising peanut (25), corn (45) and soil (15), 106 strains were isolated. Identification of strains by colony morphology and aflatoxin production found all Vietnamese strains to be A. flavus with no A. parasiticus isolated. A. flavus was present in 36.0% of peanut samples, 31.1% of corn samples, 27.3% of farmed soil samples and was not found in virgin soil samples. Twenty-five per cent of the strains produced aflatoxins. Microsatellite analysis revealed a high level of genetic diversity in the Vietnamese A. flavus population. Clustering, based on microsatellite genotype, was unrelated to aflatoxin production, geographic origin or substrate origin.     

Isolation and characterization of <Emphasis Type="Italic">Azotobacter</Emphasis> and <Emphasis Type="Italic">Azospirillum</Emphasis> strains from the sugarcane rhizosphere

Bacteria with the ability to grow on nitrogen-free media and with nitrogenase activity under aerobic or microaerobic conditions were isolated from sugarcane roots collected from four different agricultural locations in Granada (Spain). Isolates were Gram negative rods and were identified as Azotobacter chroococcum and Azospirillum brasilense. Our results suggest that Azotobacter isolates do not have a particular affinity for sugarcane rhizospheres and that, on the contrary, Azospirillum isolates show specific association and perhaps endophytic colonization of sugarcane. However, obligate endophytes (Gluconacetobacter diazotrophicus) were not found in the apoplastic fluid of the stems and macerates extracts of sugarcane tissues with the procedure applied. Population of this microorganism might be in low number in the Spanish sugarcane varieties studied which is also discussed.     

Isolation and characterization of the <Emphasis Type="Italic">Larix gmelinii </Emphasis><Emphasis Type="Italic">ANGUSTIFOLIA</Emphasis> (<Emphasis Type="Italic">LgAN</Emphasis>) gene

ANGUSTIFOLIA (AN), a plant homolog of C-terminal binding protein, controls the polar elongation of leaf cells and the trichome-branching pattern in Arabidopsis thaliana. In the present study, degenerate PCR was used to isolate an ortholog of AN, referred to as LgAN, from larch (Larix gmelinii). The LgAN cDNA is predicted to encode a protein of 646 amino acids that shows striking sequence similarity to AN proteins from other plants. The predicted amino acid sequence has a conserved NAD-dependent 2-hydroxy acid dehydrogenase (D2-HDH) motif and a plant AN-specific LxCxE/D motif at its N-terminus, as well as a plant-specific long C-terminal region. The LgAN gene is a single-copy gene that is expressed in all larch tissues. Expression of the LgAN cDNA rescued the leaf width and trichome-branching pattern defects in the angustifolia-1 (an-1) mutant of Arabidopsis, showing that the LgAN gene has effects complementary to those of AN. These results suggest that the LgAN gene has the same function as the AN gene.     

Coinfection with <Emphasis Type="Italic">Lichtheimia corymbifera</Emphasis> and <Emphasis Type="Italic">Aspergillus flavus</Emphasis> in an Immune-Competent Patient Mimicking as Pulmonary-Renal Syndrome

Lichtheimia corymbifera and Aspergillus flavus pulmonary coinfection has been rarely reported in immune-competent patients. We report case of a young male who presented with clinical features of pulmonary-renal syndrome and was later diagnosed to have bilateral polymicrobial fungal lung infection.     

Amino acid supplementation reveals differential regulation of aflatoxin biosynthesis in <Emphasis Type="Italic">Aspergillus flavus</Emphasis> NRRL 3357 and <Emphasis Type="Italic">Aspergillus parasiticus</Emphasis> SRRC 143

Aflatoxins are toxic and carcinogenic secondary metabolites produced by the fungi Aspergillus flavus and Aspergillus parasiticus. To better understand the molecular mechanisms that regulate aflatoxin production, the biosynthesis of the toxin in A. flavus and A. parasticus grown in yeast extract sucrose media supplemented with 50 mM tryptophan (Trp) were examined. Aspergillus flavus grown in the presence of 50 mM tryptophan was found to have significantly reduced aflatoxin B₁ and B₂ biosynthesis, while A. parasiticus cultures had significantly increased B₁ and G₁ biosynthesis. Microarray analysis of RNA extracted from fungi grown under these conditions revealed 77 genes that are expressed significantly different between A. flavus and A. parasiticus, including the aflatoxin biosynthetic genes aflD (nor-1), aflE (norA), and aflO (omtB). It is clear that the regulatory mechanisms of aflatoxin biosynthesis in response to Trp in A. flavus and A. parasiticus are different. These candidate genes may serve as regulatory factors of aflatoxin biosynthesis.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Isolation and characterization of <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">polyfermenticus</Emphasis> isolated from <Emphasis Type="Italic">Meju,</Emphasis> Korean soybean fermentation starter

Bacteria of the Bacillus species have been reported as an important microorganism in fermented soybean products. In the present study, thirty Bacillus isolates were screened from Meju, a Korean soybean fermentation starter. The comparative analysis of 16S rDNA sequences, 16S-23S internal transcribed spacer sequences, phenotypic, and biochemical characterizations revealed three phylogenetically distinct groups namely Bacillus atrophaeus, Bacillus polyfermenticus and Bacillus subtilis. The isolates were assayed for poly-γ-glutamate production and fibrinolytic activity. Among the isolates, B. polyfermenticus exhibited maximum poly-γ-glutamate production and fibrinolytic activity. Moreover, the soybean products fermented by B. polyfermenticus have increased the time taken for coagulation and hemorrhage in mice. The results of the present study clearly indicate the functional role of B. polyfermenticus in fermented soybean products.     

A Review Molecular Typing Methods for <Emphasis Type="Italic">Aspergillus flavus</Emphasis> Isolates

Aspergillus flavus is the second most important Aspergillus species causing human infections. The importance of this fungus increases in regions with a dry and hot climate. Small phylogenetic studies in Aspergillus flavus indicate that the morphological species contains several genetically isolated species. Different genotyping methods have been developed and employed in order to better understand the genetic and epidemiological relationships between environmental and clinical isolates. Understanding pathogen distribution and relatedness is essential for determining the epidemiology of nosocomial infections and aiding in the design of rational pathogen control methods. Typing techniques can also give us a deeper understanding of the colonization pattern in patients. Most of these studies focused on Aspergillus fumigatus because it is medically the most isolated species. To date, there has not been any publication exclusively reviewing the molecular typing techniques for Aspergillus flavus in the literature. This article reviews all these different available methods for this organism.     

Prevalence,intensity and associated factor analysis of <Emphasis Type="Italic">Tropilaelaps</Emphasis><Emphasis Type="Italic">mercedesae</Emphasis> infesting <Emphasis Type="Italic">Apis</Emphasis><Emphasis Type="Italic">mellifera</Emphasis> in China

Tropilaelaps mercedesae is a serious ectoparasite of Apis mellifera in China. The aim of this study was to investigate the infestation rates and intensity of T. mercedesae in A. mellifera in China, and to explore the relative importance of climate, district, management practices and beekeeper characteristics that are assumed to be associated with the intensity of T. mercedesae. Of the 410 participating apiaries, 379 apiaries were included in analyses of seasonal infestation rates and 352 apiaries were included in multivariable regression analysis. The highest infestation rate (86.3%) of T. mercedesae was encountered in autumn, followed by summer (66.5%), spring (17.2%) and winter (14.8%). In autumn, 28.9% (93) of the infested apiaries were in the north (including the northeast and northwest of China), 71.1% (229) were in the central and south (including east, southeast and southwest China), and 306 apiaries (82.9%) were co-infested by both T. mercedesae and Varroa. Multivariable regression analysis showed that geographical location, season, royal jelly collection and Varroa infestation were the factors that influence the intensity of T. mercedesae. The influence of beekeeper’s education, time of beekeeping, operation size, and hive migration on the intensity of T. mercedesa was not statistically significant. This study provided information about the establishment of the linkage of the environment and the parasite and could lead to better timing and methods of control.     

Elucidation of <Emphasis Type="Italic">veA</Emphasis>-dependent genes associated with aflatoxin and sclerotial production in <Emphasis Type="Italic">Aspergillus flavus</Emphasis> by functional genomics

The aflatoxin-producing fungi, Aspergillus flavus and A. parasiticus, form structures called sclerotia that allow for survival under adverse conditions. Deletion of the veA gene in A. flavus and A. parasiticus blocks production of aflatoxin as well as sclerotial formation. We used microarray technology to identify genes differentially expressed in wild-type veA and veA mutant strains that could be involved in aflatoxin production and sclerotial development in A. flavus. The DNA microarray analysis revealed 684 genes whose expression changed significantly over time; 136 of these were differentially expressed between the two strains including 27 genes that demonstrated a significant difference in expression both between strains and over time. A group of 115 genes showed greater expression in the wild-type than in the veA mutant strain. We identified a subgroup of veA-dependent genes that exhibited time-dependent expression profiles similar to those of known aflatoxin biosynthetic genes or that were candidates for involvement in sclerotial production in the wild type.     

Characterization of aflatoxigenic and non-aflatoxigenic <Emphasis Type="Italic">Aspergillus flavus</Emphasis> isolates from pistachio

Pistachio is a popular snack food. Aflatoxin contamination of pistachio nuts is a serious problem for many producing countries. The development of biological control methods based on ecological parameters is an environmentally friendly approach. Thirty-eight Aspergillus flavus isolates collected from a pistachio orchard in California (CA) were analyzed for production of aflatoxin (AF), cyclopiazonic acid (CPA), vegetative compatibility groups (VCGs), and mating types. All aflatoxigenic isolates produced both AFB₁ and CPA. The most toxigenic one was CA28 which produced 164 μg AFB₁ per 5 ml PDA fungal culture and small sclerotia (S strain, sclertoium size less than 400 μm). The other aflatoxigenic strains produce AFB₁ ranging from 1.2 μg to 80 μg per 5 ml fungal culture. Twenty-one percent of the CA isolates produced AFB₁, 84% produced CPA and half formed sclerotia on at least one of three tested media. The 38 CA isolates formed 26 VCGs, 6 of which had two or more isolates and 20 contained single isolates. The S strain isolates belong to 4 different VCGs. Genomic profiling by a retrotransposon DNA probe revealed fingerprint patterns that were highly polymorphic. The predicted VCGs (Pred-VCGs) based on a similarity coefficient >80% matched the VCGs of multiple isolates determined by complementation. All isolates within a VCG had the same mating-type gene of either MAT1-1 or MAT1-2. Uncorrected and VCG-corrected MAT1-1 and MAT1-2 among the isolates were equally distributed.     

Isolation and characterization of melanin pigment from <Emphasis Type="Italic">Pleurotus cystidiosus</Emphasis> (telomorph of <Emphasis Type="Italic">Antromycopsis</Emphasis><Emphasis Type="Italic">macrocarpa</Emphasis>)

Melanins are enigmatic pigments that are produced by a wide variety of microorganisms including several species of bacteria and fungi. For more than 40 years, fungi have been known to produce pigments called melanins. Melanin pigment production by mushrooms was not intensively studied. The present study was carried out on isolation and characterization of melanin from an edible mushroom Pleurotus cystidiosus var. formosensis. The mushroom produced dark mucous mass of hyaline arthrospores on mycelium. The coremia exclusively produced dikaryotic arthrospores with the remnant of a clamp connection. Continuous cell extension and division in the coremium stipe supplied cells for arthroconidiation at the coremium apex, which is surrounded by a liquid droplet (coremioliquid). The black coloured coremea (conidia) were produced by Antromycopsis macrocarpa (anamorph of P. cystidiosus) when cultured on potato dextrose agar medium. The agar plate was incubated at continuous light illumination for high amount of pigment (coremea) production. The slimy layer of the coremea was extracted and partially purified by alkaline and acid treatment. The black pigment was confirmed as melanin based on UV, IR and EPR spectra apart from chemical analysis. This is the first report on characterization of melanin obtained from Pleurotus cystidiosus var. formosensis.     

<Emphasis Type="Italic">Aspergillus flavus</Emphasis> hydrolases: their roles in pathogenesis and substrate utilization

Aspergillus flavus is a fungus that principally obtains resources for growth in a saprophytic mode. Yet, it also possesses the characteristics of an opportunistic pathogen with a wide, non-specific host range (plants, animals, and insects). It has attained a high level of agricultural significance due to production of the carcinogen aflatoxin, which significantly reduces the value of contaminated crops. To access a large variety of nutrient substrates and penetrate host tissues, A. flavus possesses the capacity to produce numerous extracellular hydrolases. Most work on A. flavus hydrolases has focused on the serine and metalloproteinases, pectinase P2c, and amylase. Many hydrolases are presumed to function in polymer degradation and nutrient capture, but the regulation of hydrolase secretion is complex and substrate dependent. Proteinases are employed not only to help access protein substrates, such as elastin that is found in mammals and insects, but may also play roles in fungal defense and virulence. Secretion of the endopolygalacturonase P2c is strongly correlated with isolate virulence (against plants) and maceration of cotton boll tissues. In some hosts, secretion of α-amylase is critical for starch digestion and may play a critical role in induction of aflatoxin biosynthesis. Despite a significant body of work, much remains to be learned about hydrolase production and utilization by A. flavus. This information may be critical for the formulation of successful strategies to control aflatoxin contamination in affected commodities.     

Biological Interactions to Select Biocontrol Agents Against Toxigenic Strains of <Emphasis Type="Italic">Aspergillus flavus</Emphasis> and <Emphasis Type="Italic">Fusarium verticillioides</Emphasis> from Maize

Biological control represent an alternative to the use of pesticides in crop protection. A key to progress in biological control to protect maize against Fusarium verticillioides and Aspergillus flavus maize pathogens are, to select in vitro, the best agent to be applied in the field. The aim of this study was to examine the antagonistic activity of bacterial and yeast isolates against F.verticillioides and A. flavus toxigenic strains. The first study showed the impact of Bacillus amyloliquefaciens BA-S13, Microbacterium oleovorans DMS 16091, Enterobacter hormomaechei EM-562T, and Kluyveromyces spp. L14 and L16 isolates on mycelial growth of two strains of A. flavus MPVPA 2092, 2094 and three strains of F. verticillioides MPVPA 285, 289, and 294 on 3% maize meal extract agar at different water activities (0.99, 0.97, 0.95, and 0.93). From this first assay antagonistics isolates M. oleovorans, B. amyloliquefaciens and Kluyveromyces sp. (L16) produced an increase of lag phase of growth and decreased a growth rate of all fungal strains. These isolates were selected for futher studies. In vitro non-rhizospheric maize soil (centrally and sprayed inoculated) and in vitro maize (ears apex and base inoculated) were treated with antagonistics and pathogenic strains alone in co-inoculated cultures. Bacillus amyloliquefaciens significantly reduced F. verticillioides and A. flavus count in maize soil inoculated centrally. Kluyveromyces sp. L16 reduced F. verticillioides and A. flavus count in maize soil inoculated by spray. Kluyveromyces sp. L16 was the most effective treatment limiting percent infections by F. verticillioides on the maize ears.     

Quantitative study of interactions between <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and <Emphasis Type="Italic">Oenococcus </Emphasis><Emphasis Type="Italic">oeni</Emphasis> strains

This study examines the interactions that occur between Saccharomyces cerevisiae and Oenococcus oeni strains during the process of winemaking. Various yeast/bacteria pairs were studied by applying a sequential fermentation strategy which simulated the natural winemaking process. First, four yeast strains were tested in the presence of one bacterial strain leading to the inhibition of the bacterial component. The extent of inhibition varied widely from one pair to another and closely depended on the specific yeast strain chosen. Inhibition was correlated to weak bacterial growth rather than a reduction in the bacterial malolactic activity. Three of the four yeast strains were then grown with another bacteria strain. Contrary to the first results, this led to the bacterial stimulation, thus highlighting the importance of the bacteria strain. The biochemical profile of the four yeast fermented media exhibited slight variations in ethanol, SO(2) and fatty acids produced as well as assimilable consumed nitrogen. These parameters were not the only factors responsible for the malolactic fermentation inhibition observed with the first bacteria strain. The stimulation of the second has not been reported before in such conditions and remains unexplained.