Effect of Additives on the Production, Viability and Virulence of Blastospores of Metarhizium anisopliae

Studies on the blastospore production of Metarhizium anisopliae var. anisopliae were conducted in Adamek's medium used as a standard, enriched with lecithin, collagen, lactic acid or polyethylene glycol 200 (PEG 200) to increase spore yield and suppress mycelial pellet formation. The addition of 5% lecithin resulted in a significant 10-fold increase in spore yield up to 1.9 108 blastospores/ml compared with 1.9 107 spores/ml in the standard medium. Collagen (3%) increased the number of blastospores 3.7-fold, and lactic acid (1.5%) two-fold. A reduction of mycelial pellet formation in favour of spore production was noted with each additive. The viability of blastospores at 40IC from media with lecithin, collagen and lactic acid suspended in 25% Ringer's solution was comparable to that of spores produced in the standard medium. Striking differences were noticed in the viability of spores produced with 5% PEG 200 in standard medium. The half-life of blastospores produced in standard medium suspended in sunflower oil was 33.6 h and that of 5% PEG 200 spores only 25.2 h. In bioassays, the virulence of spores produced in standard medium to which 3% lecithin, 3% collagen, 1.5% lactic acid or 5% PEG 200 had been added was tested against third-instar nymphs of Locusta migratoria migratorioides (R. & F.). The median lethal time and the mortality of L. migratoria achieved with blastospores produced with 3% lecithin (5.7 days, 99%) was comparable to that of blastospores from standard medium (5.1 days, 98%). The virulence of blastospores from all other media with additives was significantly reduced.     

Substrate-induced lipase gene expression and aflatoxin production in Aspergillus parasiticus and Aspergillus flavus

AIMS: To establish a relationship between lipase gene expression and aflatoxin production by cloning the lipA gene and studying its expression pattern in several aflatoxigenic and nontoxigenic isolates of Aspergillus flavus and A. parasiticus. METHODS AND RESULTS: We have cloned a gene, lipA, that encodes a lipase involved in the breakdown of lipids from aflatoxin-producing A. flavus, A. parasiticus and two nonaflatoxigenic A. flavus isolates, wool-1 and wool-2. The lipA gene was transcribed under diverse media conditions, however, no mature mRNA was detected unless the growth medium was supplemented with 0.5% soya bean or peanut oil or the fungus was grown in lipid-rich medium such as coconut medium. The expression of the lipase gene (mature mRNA) under substrate-induced conditions correlated well with aflatoxin production in aflatoxigenic species A. flavus (SRRC 1007) and A. parasiticus (SRRC 143). CONCLUSIONS: Substrate-induced lipase gene expression might be indirectly related to aflatoxin formation by providing the basic building block 'acetate' for aflatoxin synthesis. No direct relationship between lipid metabolism and aflatoxin production can be ascertained, however, lipase gene expression correlates well with aflatoxin formation. SIGNIFICANCE AND IMPACT OF THE STUDY: Lipid substrate induces and promotes aflatoxin formation. It gives insight into genetic and biochemical aspects of aflatoxin formation.     

Cloning of the afl-2 gene involved in aflatoxin biosynthesis from Aspergillus flavus.

Aflatoxins are extremely potent carcinogens produced by Aspergillus flavus and Aspergillus parasiticus. Cloning of genes in the aflatoxin pathway provides a specific approach to understanding the regulation of aflatoxin biosynthesis and, subsequently, to the control of aflatoxin contamination of food and feed. This paper reports the isolation of a gene involved in aflatoxin biosynthesis by complementation of an aflatoxin-nonproducing mutant with a wild-type genomic cosmid library of A. flavus. Strain 650-33, blocked in aflatoxin biosynthesis at the afl-2 allele, was complemented by a 32-kb cosmid clone (B9), resulting in the production of aflatoxin. The onset and profile of aflatoxin accumulation was similar for the transformed strain and the wild-type strain (NRRL 3357) of the fungus, indicating that the integrated gene is under the same control as in wild-type strains. Complementation analyses with DNA fragments from B9 indicated that the gene resides within a 2.2-kb fragment. Because this gene complements the mutated afl-2 allele, it was designated afl-2. Genetic evidence obtained from a double mutant showed that afl-2 is involved in aflatoxin biosynthesis before the formation of norsolorinic acid, the first stable intermediate identified in the pathway. Further, metabolite feeding studies with the mutant, transformed, and wild-type cultures and enzymatic activity measurements in cell extracts of these cultures suggest that afl-2 regulates gene expression or the activity of other aflatoxin pathway enzymes. This is the first reported isolation of a gene for aflatoxin biosynthesis in A. flavus.     

In vivo development of the entomogeneous hyphomycetePaecilomyces farinosus in hostSpodoptera exigua (beet armyworm) larvae

The in vivo development of the entomogenous hyphomycetePaecilomyces farinosus inSpodoptera exigua (beet armyworm) larvae was examined using light and electron microscopic techniques. Blastospores injected into larval hemocoels (500 blastospores/larva) were immediately ingested by phagocytic hemocytes, and no fungal cells were detected in the hemolymph until 36 h post-injection. As indicated by immunocytochemical methods, the in vivo-produced blastosopres, in contrast to in vitro blastospores, lacked a galacto-mannan surface layer required for opsonization by aS. exigua humoral lectin. Therefore, these in vivo cells were not recognized by phagocytic granulocytes and were freely-circulating in the hemolymph. Hyphae differentiating from the blastospores were recognized by the hemocytes and induced formation of multicellular hemocytic nodules. By 72 h post-injection, mycelia were observed emerging from the nodules and by 96 h, larvae had become mummified due to extensive proliferation of the fungus throughout host tissues. Neither phagocytosis of the initially injected in vitro-produced blastospores nor nodule formation around hyphal cells later in the infection process was effective in stopping fungal growth. The in vivo development ofP. farinosus was similar to that of another hyphomycete,Beauveria bassiana except that in the latter case, extensive nodule formation was inhibited by the production of fungal metabolites.     

Coconut as a Medium for the Experimental Production of Aflatoxin

Fresh, grated coconut has been found to be an excellent medium for aflatoxin production by Aspergillus flavus. Under optimal conditions, yields of 8 mg of total aflatoxin per g of substrate were obtained. Continuous agitation of the growth medium under moist conditions at 24 C produced highest yields. Aflatoxin was assayed both biologically and chromatographically. The aflatoxin content of cultures varied biphasically with the duration of incubation. It is suggested that this pattern could result from the sequential operation of factors promoting aflatoxin formation on the one hand and a detoxifying mechanism on the other.     

Evaluation of the antigen from chlamydospores of Candida albicans in the serodiagnosis of candidiasis

Antigens have been prepared from the chlamydospores and blastospores of Candida albicans and their precipitin patterns were analysed by two-dimensional immunoelectrophoresis using specific antisera.The two antigens were used in routine serological tests of patients suffering from candidiasis. On double-diffusion tests for the detection of circulating antibodies of Candida albicans, the antigen from chlamydospores displays precipitin lines that differ in number and intensity from those obtained with the antigen from blastospores. The results are briefly discussed in the framework of C. albicans antigen standardization.     

Some high-performance liquid-chromatographic studies of the metabolism of aflatoxins by rat liver microsomal preparations.

The metabolism of aflatoxin B1 in vitro was examined in rat liver microsomal preparations. 2. H.p.l.c. (high-performance liquid-chromatographic) systems were used. A silica column was used to separate non-polar metabolites. A system utilizing a reversed-phase column which separates both poar and non-polar metabolites was also developed. 3. The principal metabolites of aflatoxin B1 found were aflatoxin M1, aflatoxin Q1 and a compound which co-chromatographed with a degradation product of aflatoxin B1 2,3-dihydrodiol. 4. The time course of metabolism of aflatoxin B1 by microsomal preparations isolated from control and phenobarbitone-pretreated rats was examined. The rate and extent of metabolism was greater with microsomal preparations from the latter. The formation of aflatoxin Q1 was enhanced 4--5-fold by phenobarbitone pretreatment, whereas the production of aflatoxin M1 was only increased 1--2-fold. The formation of the degradation product of aflatoxin B1 2,3-dihydrodiol was increased 4--5-fold by the pretreatment with phenobarbitone. 5. The microsomal metabolism of aflatoxins M1, P1 and Q1 was examined. Aflatoxin M1 apparently underwent very limited microsomal metabolism to more polar compounds. Aflatoxin P1 was not metabolized. The situation with aflatoxin Q1 was complicated in that it was metabolized in the absence of NADPH to an unidentified metabolite. Aflatoxin B1 appeared as a metabolite of aflatoxin Q1 only when NADPH was present, and the formation of more polar metabolites was also then observed.     

碳源对玫烟色虫草菌丝生长、芽生孢子产生及其耐热性的影响

提高虫生真菌孢子应对热胁迫的能力是生防菌应用研究的关键,为研究菌丝培养阶段碳源对玫烟色虫草Cordyceps fumosorosea IF-1106耐热性的影响,选择了麦芽糖、可溶性淀粉、蔗糖、葡萄糖、果糖、海藻糖为碳源的培养基对玫烟色虫草IF-1106进行液体培养,评估了不同碳源条件下菌丝的生长、产孢及所产芽生孢子的耐热性。结果表明,在菌株培养阶段,培养基中碳源的种类及浓度对菌丝产量、产孢量及所产芽生孢子的耐热性有显著影响,其中蔗糖为碳源时,所产芽生孢子的耐热性强,45 ℃热胁迫条件下LT₅₀为1.65 h;蔗糖浓度为40 g/L时,可产生大量耐热芽生孢子,液体培养3 d后产孢量可达3.43×10⁷个孢子/mL。为探索不同培养条件下所产芽生孢子耐热性不同的原因,提取了孢子内的海藻糖并采用离子色谱法对其进行了定量分析,发现耐热性高的芽生孢子胞内海藻糖含量普遍较低,可见海藻糖是与芽生孢子耐热性密切相关的内源物质。综上所述,选择适宜的培养基是调控孢子耐热性的有效途径,本研究为生产高耐热的玫烟色虫草生防制剂提供了有益的指导。     

The formation of protoplasts from Beauveria bassiana

Summary Beauveria bassiana protoplast formation from blastospores, conidia and mycelia was studied. The method of protoplast formation involves preincubation of the fungal cells with dithiothreitol and subsequent treatment with an enzyme mixture consisting of: cellulase, chitinase, -glucuronidase and lysozyme. Using this procedure protoplasts were formed from blastospores and mycelia but not conidia. Formation of protoplasts from 24 hour old mycelia was 100% efficient using the above conditions. A number of ionic and osmotic protoplast stabilizing agents were tested. Ammonium sulfate was shown to be the stabilizer of choice. Protoplasts were stable when stored at 4° C with a loss of only 17% in 6 days. We suggest that this procedure of protoplast production will allow a gentler method for the extraction and isolation of intact high molecular weight DNA from B. bassiana.     

Factors influencing the inhibition of aflatoxin production in corn by Aspergillus niger

Aspergillus niger, a mold commonly associated with Aspergillus flavus in damaged corn, interferes with the production of aflatoxin when grown with A. flavus on autoclaved corn. The pH of corn-meal disks was adjusted using NaOH-HCl, citric acid-sodium citrate, or a water extract of A. niger fermented corn. Aflatoxin formation was completely inhibited below pH 2.8-3.0, irrespective of the system used for pH adjustment. When grown in association with A. flavus NRRL 6432 on autoclaved corn kernels, A. niger NRRL 6411 lowered substrate pH sufficiently to suppress aflatoxin production. The biodegradation of aflatoxin B1 or its conversion to aflatoxin B2a were eliminated as potential mechanisms by which A. niger reduces aflatoxin contamination. A water extract of corn kernels fermented with A. niger caused an additional inhibition of aflatoxin formation apart from the effects of pH.     

Inhibition of aflatoxin biosynthesis by tolnaftate.

Tolnaftate [2-napthyl-N-methyl-N-(m-tolyl)thionocarbamate], an antifungal drug, is widely used to control superficial fungal infections in humans and other animals. In this study the effect of tolnaftate on aflatoxin biosynthesis by Aspergillus parasiticus NRRL 3240 was investigated. Tolnaftate changed the morphology of A. parasiticus to yeastlike forms and inhibited aflatoxin formation. The formation of aflatoxin G was blocked considerably, indicating a metabolic block in the conversion of aflatoxin B to aflatoxin G. The incorporation of [1-14C]acetate into aflatoxin was significantly inhibited at a concentration of 1 mM tolnaftate. The presence of zinc in the resuspension buffer resulted in reversal of the tolnaftate-induced inhibition of aflatoxin G1 biosynthesis.     

Calmodulin mediated activation of acetyl-CoA carboxylase during aflatoxin production by Aspergillus parasiticus

The relevance of Ca2+-calmodulin-mediated processes in channelling acetate for aflatoxin formation was investigated by studying the influence of trifluoperazine (an anticalmodulin agent) on [14C]-acetate incorporation and activity of acetyl-CoA carboxylase in Aspergillus parasiticus NRRL 2999. Culturing the organism in presence of 0.14 mmol l-1 trifluoperazine resulted in 55% decrease of [14C]-acetate incorporation into aflatoxin B1, along with an 80% decrease in acetyl-CoA carboxylase activity at periods corresponding to maximal aflatoxin production. Concomitant decrement (35%) in the activity of glucose-6-phosphate dehydrogenase indicated decreased availability of reduction potential (NADPH) required for aflatoxin biosynthesis. The ability of calmodulin to activate and trifluoperazine to inhibit acetyl-CoA carboxylase activity in a dose-dependent manner was also noted under in vitro conditions. The combined results suggest calmodulin-mediated activation of acetyl-CoA carboxylase as an important event for aflatoxin production.     

Purified moniliformin does not affect the force or rate of contraction of isolated guinea pig atria

Aspergillus flavus Link ex Fries and A. parasiticus Speare can invade peanut kernels and under certain environmental conditions produce unacceptable levels of the mycotoxin aflatoxin. A concerted effort is underway to reduce aflatoxin contamination in peanut and peanut products. A potentially effective method of control in peanut is the discovery and use of genes for resistance to either fungal invasion or aflatoxin formation. The objective of the present experimental study was to develop an effective and efficient procedure for screening individual plants or pods of single plants for resistance to invasion by the aflatoxigenic fungi and subsequent aflatoxin production. Methods of obtaining adequate drought-stress and fungal infection were developed through this series of experiments. By completely isolating the pods from the root zone and imposing drought-stress only on pegs and pods, high levels of fungal infection were observed. High amounts of preharvest aflatoxin accumulation were also produced by completely isolating the pods from the root zone. Mid-bloom inoculation with A. parasiticus-contaminated cracked corn and drought-stress periods of 40 to 60 days were the most effective procedures. This technique was used to assess peanut genotypes previously identified as being partially resistant to A. parasiticus infection or aflatoxin contamination, and segregating populations from four crosses. Variability in aflatoxin contamination was found among the 11 genotypes evaluated, however, none were significantly lower than the standard cultivars. Broad-sense heritability of four crosses was estimated through evaluation of seed from individual plants in the F₂ generation. The heritability estimates of crosses GFA-2 × NC-V11 and Tifton-8 × NC-V11 were 0.46 and 0.29, respectively, but mean aflatoxin contamination levels were high (73,295 and 27,305 ppb). This greenhouse screening method could be an effective tool when genes for superior aflatoxin resistance are identified.Cooperative investigation of the USDA-ARS and the University of Georgia, College of Agriculture.     

Enzymatic Formation of G-Group Aflatoxins and Biosynthetic Relationship between G- and B-Group Aflatoxins

We detected biosynthetic activity for aflatoxins G₁ and G₂ in cell extracts of Aspergillus parasiticus NIAH-26. We found that in the presence of NADPH, aflatoxins G₁ and G₂ were produced from O-methylsterigmatocystin and dihydro-O-methylsterigmatocystin, respectively. No G-group aflatoxins were produced from aflatoxin B₁, aflatoxin B₂, 5-methoxysterigmatocystin, dimethoxysterigmatocystin, or sterigmatin, confirming that B-group aflatoxins are not the precursors of G-group aflatoxins and that G- and B-group aflatoxins are independently produced from the same substrates (O-methylsterigmatocystin and dihydro-O-methylsterigmatocystin). In competition experiments in which the cell-free system was used, formation of aflatoxin G₂ from dihydro-O-methylsterigmatocystin was suppressed when O-methylsterigmatocystin was added to the reaction mixture, whereas aflatoxin G₁ was newly formed. This result indicates that the same enzymes can catalyze the formation of aflatoxins G₁ and G₂. Inhibition of G-group aflatoxin formation by methyrapone, SKF-525A, or imidazole indicated that a cytochrome P-450 monooxygenase may be involved in the formation of G-group aflatoxins. Both the microsome fraction and a cytosol protein with a native mass of 220 kDa were necessary for the formation of G-group aflatoxins. Due to instability of the microsome fraction, G-group aflatoxin formation was less stable than B-group aflatoxin formation. The ordA gene product, which may catalyze the formation of B-group aflatoxins, also may be required for G-group aflatoxin biosynthesis. We concluded that at least three reactions, catalyzed by the ordA gene product, an unstable microsome enzyme, and a 220-kDa cytosol protein, are involved in the enzymatic formation of G-group aflatoxins from either O-methylsterigmatocystin or dihydro-O-methylsterigmatocystin.     

Direct visual detection of aflatoxin synthesis by minicolonies of Aspergillus species

Single-spore colonies of Aspergillus flavus and Aspergillus parasiticus, grown for 4 to 5 days at 25 degrees C on a coconut extract agar containing sodium desoxycholate as a growth inhibitor, produced aflatoxin, readily detectable as blue fluorescent zones under long-wave (365 nm) UV light. Over 100 colonies per standard petri dish were scored for aflatoxin production by this procedure. Progeny from some strains remained consistently stable for toxin production after repeated subculture, whereas instability for toxin synthesis was revealed among progeny from other strains. Spore color markers were used to rule out cross-contamination in monitoring strains. A yellow-spored and nontoxigenic strain of A. flavus, reported previously to produce aflatoxin in response to cycloheximide treatment, proved to be toxin negative even after repeated exposure to cycloheximide. Extended series of progeny from another strain of A. flavus and from a strain of A. parasiticus were each compared by this plating procedure and by fluorometric analysis for aflatoxin when grown in a coconut extract broth. Both of these strains showed variation for toxin synthesis among their respective progeny, and specific progeny showed a good correlation for aflatoxin synthesis when examined by the two procedures.     

Direct visual detection of aflatoxin synthesis by minicolonies of Aspergillus species.

Single-spore colonies of Aspergillus flavus and Aspergillus parasiticus, grown for 4 to 5 days at 25 degrees C on a coconut extract agar containing sodium desoxycholate as a growth inhibitor, produced aflatoxin, readily detectable as blue fluorescent zones under long-wave (365 nm) UV light. Over 100 colonies per standard petri dish were scored for aflatoxin production by this procedure. Progeny from some strains remained consistently stable for toxin production after repeated subculture, whereas instability for toxin synthesis was revealed among progeny from other strains. Spore color markers were used to rule out cross-contamination in monitoring strains. A yellow-spored and nontoxigenic strain of A. flavus, reported previously to produce aflatoxin in response to cycloheximide treatment, proved to be toxin negative even after repeated exposure to cycloheximide. Extended series of progeny from another strain of A. flavus and from a strain of A. parasiticus were each compared by this plating procedure and by fluorometric analysis for aflatoxin when grown in a coconut extract broth. Both of these strains showed variation for toxin synthesis among their respective progeny, and specific progeny showed a good correlation for aflatoxin synthesis when examined by the two procedures.     

Ultrastructural investigations of the formation of Candida albicans germ tubes and septa.

Germ tubes, blastospores and pseudomycelia of Candida albicans were studied under the electron microscope. Both germ tube and blastospore formation begin with comparable changes in the cell wall of the mother cell. The major part of the germ tube wall consists of the fourth of the five layers forming the C. albicans cell wall. Germ tubes are precursors of mycelia which can ramify. Septa and septal pores can be demonstrated in later phases. Pseudomycelia can be clearly discerned by means of transmission electron microscopy.     

Differential susceptibility of blastospores and aerial conidia of entomopathogenic fungi to heat and UV-B stresses

We investigated the comparative susceptibility to heat and UV-B radiation of blastospores and aerial conidia of Metarhizium spp. (Metarhizium robertsii IP 146, Metarhizium anisopliae s.l. IP 363 and Metarhizium acridum ARSEF 324) and Beauveria bassiana s.l. (IP 361 and CG 307). Conidia and blastospores were produced in solid or liquid Adámek-modified medium, respectively, and then exposed to heat (45 ± 0.2 °C) in a range of 0 (control) to 360 min; the susceptibility of fungal propagules to heat exposures was assessed to express relative viability. Similarly, both propagules of each isolate were also exposed to a range of 0 (control) to 8.1 kJ m⁻² under artificial UV-B radiation. Our results showed that fungal isolates, propagule types and exposure time or dose of the stressor source play critical roles in fungal survival challenged with UV-B and heat. Conidia of ARSEF 324, IP 363, IP 146 and IP 361 exposed to heat survived significantly longer than their blastospores, except for blastospores of CG 307. Conidia and blastospores of IP 146 and IP 363 were equally tolerant to UV-B radiation. We claim that blastospores of certain isolates may be promising candidates to control arthropod pests in regions where heat and UV-B are limiting environmental factors.     

Effect of peanut tannin extracts on growth of Aspergillus parasiticus and aflatoxin production

Twenty-three peanut (Arachis hypogaea L.) genotypes were evaluated for kernel resistance to Aspergillus parasiticus Spear. colonization and aflatoxin contamination when incubated under high relative humidity. Also, tannin-containing extracts from kernel coats (testae) and cotyledons of these genotypes were prepared and tested for their effect on A. parasiticus growth and aflatoxin production in vitro. The lowest degree of colonization, less than 30% was noted in kernels from the genotypes, Toalson x UF 73-4022 (selections TX-798731 and TX-798736), A72118, SN 55-437, PI337409, and Florunner. Genotypes with low levels of colonization also had the lowest aflatoxin contamination. The coefficient of correlation between infection frequency and aflatoxin contamination was 0.66. Higher levels of tannins were detected in the testae (23.9–97.2 mg g tissue) compared to the cotyledons (0.17–0.82 mg g tissue). Some of the methanol-extracted and water-soluble tannin extracts from testae and cotyledons, when incorporated in yeast extract sucrose liquid medium (100 mg l), significantly inhibited A. parasiticus growth and reduced the levels of aflatoxin produced. There was no overall correlation between the peanut genotypes and the influence of tannin extracts on A. parasiticus growth and aflatoxin production. However, correlations were higher for specific genotypes. For example, the coefficient of correlation between the ability of tannin extracts from testae of genotypes PI337409 and TX-798736 to inhibit aflatoxin production was 0.93 and 0.85 respectively.     

Intracellular aflatoxin B1-binding proteins in rat liver

Intracellular aflatoxin B1 binding in rat liver was studied under both in vitro and in vivo conditions. Binding in vivo appeared similar to that observed in vitro except that some covalent adduct formation was detected. Participation of previously described carcinogen-binding proteins such as the Ah receptor, h2-5S protein, 4-5S receptor for 3-methylcholanthrene and the Z-protein fraction was discounted on the grounds of competition binding studies and gel-permeation chromatography. The molecular weight of 45,000 was estimated for the major aflatoxin B1-binding component. Aflatoxin B1 co-eluted with the glutathione S-transferases during gel-permeation and separation of the various isozymes by cation-exchange chromatography indicated interactions with the YaYa and YaYc-forms. These proteins, however, account for less than 20% of the total intracellular aflatoxin binding. A protein of apparent monomeric structure appears to form the major in vitro/in vivo complex with aflatoxin B1.