Intrafungal distribution of aflatoxins among conidia and sclerotia of Aspergillus flavus and Aspergillus parasiticus

This research examines the distribution of aflatoxins among conidia and sclerotia of toxigenic strains of Aspergillus flavus Link and Aspergillus parasiticus Speare cultured on Czapek agar (21 days, 28 degrees C). Total aflatoxin levels in conidia and sclerotia varied considerably both within (intrafungal) and among strains. Aspergillus flavus NRRL 6554 accumulated the highest levels of aflatoxin (conidia: B1, 84000 ppb; G1, 566000 ppb; sclerotia: B1, 135000 ppb; G1, 968000 ppb). Substantial aflatoxin levels in conidia could place at risk those agricultural workers exposed to dust containing large numbers of A. flavus conidia. Cellular ratios of aflatoxin B1 to aflatoxin G1 were nearly identical in conidia and sclerotia even though levels of total aflatoxins in these propagule types may have differed greatly. Aflatoxin G1 was detected in sclerotia of all A. flavus strains but in the conidia of only one strain. Each of the A. parasiticus strains examined accumulated aflatoxin G1 in both sclerotia and conidia. These results are examined in the context of current evolutionary theory predicting an increase in the chemical defense systems of fungal sclerotia, propagules critical to the survival of these organisms.     

Deletion of the Delta12-oleic acid desaturase gene of a nonaflatoxigenic Aspergillus parasiticus field isolate affects conidiation and sclerotial development

AIMS: To investigate how linoleic acid affects conidial production and sclerotial development in a strictly mitotic Aspergillus parasiticus field isolate as related to improving biocompetitivity of atoxigenic Aspergillus species. METHODS AND RESULTS: We disrupted A. parasiticusDelta12-oleic acid desaturase gene (odeA) responsible for the conversion of oleic acid to linoleic acid. We examined conidiation and sclerotial development of SRRC 2043 and three isogenic mutant strains deleted for the odeA gene (DeltaodeA), either with or without supplementing linoleic acid, on one complex potato dextrose agar (PDA) medium and on two defined media: nitrate-containing Czapek agar (CZ) and Cove's ammonium medium (CVN). The DeltaodeA mutants produced less conidia than the parental strain on all media. Linoleic acid supplementation (as sodium linoleate at 0.3 and 1.2 mg ml(-1)) restored the DeltaodeA conidial production comparable to or exceeding the unsupplemented parental level, and the effect was medium dependent, with the highest increase on CVN and the least on PDA. SRRC 2043 and the DeltaodeA mutants were unable to produce sclerotia on CVN. On unsupplemented PDA and CZ, DeltaodeA sclerotial mass was comparable to that of SRRC 2043, but sclerotial number increased significantly to two- to threefold. Supplementing linoleic acid to media, in general, tended to decrease wild type and DeltaodeA sclerotial mass and sclerotial number. CONCLUSIONS: Linoleic acid stimulates conidial production but has an inhibitory effect on sclerotial development. The relationship between the two processes in A. parasiticus is complex and affected by multiple factors, such as fatty acid composition and nitrogen source. SIGNIFICANCE AND IMPACT OF STUDY: Conditions that promote sclerotial development differ from those required to promote maximum conidial production. Manipulation of content and availability of linoleic acid at different fungal growth phases might optimize conidial and sclerotial production hence increasing the efficacy of biocompetitive Aspergillus species.     

Effects of laeA deletion on Aspergillus flavus conidial development and hydrophobicity may contribute to loss of aflatoxin production

LaeA of Aspergillus nidulans is a putative methyltransferase and a component of the velvet complex; it is thought to mainly affect expression of genes required for the production of secondary metabolites. We found that although Aspergillus flavus CA14 laeA deletion mutants showed no aflatoxin production, expression of some of the early genes involved in aflatoxin formation, but not the later genes, could still be detected. The mutants grown in minimal medium supplemented with simple sugars and on some complex media exhibited altered conidial development. On potato dextrose agar (PDA) medium the deletion mutants showed reduced conidial chain elongation, increased production of conidiophores, and decreased colony hydrophobicity when compared to the parental strain. The loss of hydrophobicity and the other developmental changes in the laeA deletion mutants could affect the ability of the fungus to produce aflatoxins.     

THE EFFECT OF SOME PHYSIOLOGICAL AND ENVIRONMENTAL FACTORS ON SCLEROTIAL ASPERGILLI

Rudolph , Emanuel D. (Ohio State U., Columbus.) The effect of some physiological and environmental factors on sclerotial Aspergilli. Amer. Jour. Bot. 49(1): 71–78. Illus. 1962.—The effect of varying conditions of carbon-nitrogen balance, temperature, pH, and light upon the formation of sclerotia by 6 species of Aspergillus (A. alliaceus, A. avenaceus, A. flavus, A. quercinus, A. sclerotiorum and A. wentii) was studied. On Czapek's agar, optimal growth as well as maximum production of sclerotia and conidia took place at high sucrose and nitrate concentrations. In general, fewer sclerotia were formed with glucose than with sucrose, and very poor growth took place with lactose. Sclerotia were formed best at temperatures that were optimal or below optimal for mycelial growth. The ranges of pH through which sclerotia were formed were narrower than those through which conidia and mycelia were formed. Light had no effect upon sclerotium formation. The formation of sclerotia in A. alliaceus was found to represent the strand-type development. A number of UV-induced strains and a spontaneous mutant strain of A. alliaceus showing varying amounts of sclerotium and conidium production are characterized. It is suggested that the sclerotia in Aspergillus are sterile stromata.     

Interrelation of growth media and water activity in sclerotia characteristics of Aspergillus section Flavi

AIMS: To examine sclerotium characteristics of two Aspergillus flavus and two A. parasiticus strains at different growth media and water stress. METHODS AND RESULTS: The effects of growth media and water activity (0.999, 0.971, 0.955 and 0.937) on characteristics of sclerotia production (number, size and volume) of four isolates of Aspergillus section Flavi were examined. There was total inhibition under the driest conditions (0.955 and 0.937). When an osmotic potential of 0.971 was generated in Czapek agar (CD) and maize meal extract agar with sucrose and sodium nitrate (MMEA S/N), an increase in sclerotial size and volume was observed. The amount of sclerotia produced by cultures at 0.999 a(w) value was higher on CD. CONCLUSIONS: The data show that the sclerotia characteristics of A. flavus and A. parasiticus have been influenced by water availability and growth media composition. SIGNIFICANCE AND IMPACT OF THE STUDY: The information obtained shows that if we know the nutritional and water stress requirements for sclerotia production, it could be possible to develop effective prevention strategies to inhibit the survival of these fungi in grain.     

Effect of competition and adverse culture conditions on aflatoxin production by Aspergillus flavus through successive generations

Strains of Aspergillus flavus often degenerate with serial transfers on culture media, resulting in morphological changes and loss of aflatoxin production. However, degeneration does not readily occur in nature as indicated by the wild-type morphological characters of newly isolated strains and the high percentage of aflatoxigenic A. flavus from soil and crops in some geographic regions. In this study, three aflatoxin-producing strains of A. flavus were serially transferred using conidia for 20 generations (three independent generation lines per strain) on potato dextrose agar at 30 C. The rate of degeneration was compared to that of cultures grown in the presence of competing fungi (A. terreus, Penicillium funiculosum, and the yeast, Pichia guilliermondii) and under adverse conditions of elevated temperature, reduced water activity, low pH, and nutrient deprivation. Formation of morphological variants and the associated loss of aflatoxin production over generations varied considerably according to strain and the generation line within each strain. In the strain most sensitive to degeneration on potato dextrose agar, aflatoxin-producing ability was maintained to varying degrees under adverse culture conditions, but not when A. flavus was competing with other fungi.     

Association of aflatoxin biosynthesis and sclerotialdevelopment in Aspergillus parasiticus

Secondary metabolism in fungi is frequently associated with asexual and sexual development. Aspergillus parasiticus produces aflatoxins known to contaminate a variety of agricultural commodities. This strictly mitotic fungus, besides producing conidia asexually, produces sclerotia, structures resistant to harsh conditions and for propagation. Sclerotia are considered to be derived from the sexual structure, cleistothecia, and may represent a vestige of ascospore production. Introduction of the aflatoxin pathway-specific regulatory gene, aflR, and aflJ, which encoded a putative co-activator, into an O-methylsterigmatocystin (OMST)-accumulating strain,A. parasiticus SRRC 2043, resulted in elevated levels of accumulation of major aflatoxin precursors, including norsolorinic acid (NOR), averantin (AVN), versicolorin A (VERA) and OMST. The total amount of these aflatoxin precursors, NOR, VERA, AVN and OMST, produced by the aflR plus aflJ transformants was two to three-fold that produced by the aflR transformants. This increase indicated a synergisticeffect of aflR and aflJ on the synthesis of aflatoxin precursors. Increased production of the aflatoxin precursors was associated with progressive decrease in sclerotial size, alteration in sclerotial shape and weakening in the sclerotial structure of the transformants. The results showed that sclerotial development and aflatoxin biosynthesis are closely related. We proposed that competition for a common substrate, such as acetate, by the aflatoxin biosynthetic pathway could adversely affect sclerotial development in A. parasiticus. This revised version was published online in June 2006 with corrections to the Cover Date.     

蛋白激酶Cla4对黄曲霉生长发育、毒素合成和致病力的影响

黄曲霉Aspergillus flavus是机会性的动物和植物致病性丝状真菌,保守的PAK(p21-activated protein kinases)样蛋白激酶对信号传导、细胞周期进程和细胞形态发生具有重要作用.通过同源重组方法构建敲除突变株(ΔAflcla4),研究Aflcla4基因对黄曲霉营养生长、分生孢子产生、...     

Heterokaryosis and Alkaloid Production in Claviceps purpurea

Strain 275 FI of Claviceps purpurea, which produces large amounts of peptide alkaloids in submerged culture, is a heterokaryon; after several generations on agar media, it segregates the single components. These components (by us labeled V, C, and W) are stable and produce almost no alkaloids under described conditions of submerged culture. The mycelium of strain 275 FI consists of hyphae with multinucleate cells and does not produce conidia. Strains V, C, and W form numerous anastomoses when grown together on agar. By combining strains V and C, a heterokaryon similar to 275 FI in appearance has been obtained. This new strain produces amounts of alkaloids much larger than those produced by V and C separately or in associated submerged culture. We conclude, therefore, that in strain 275 FI the heterokaryotic condition is favorable to the production of alkaloids. Several conidiaproducing cultures of C. purpurea of various origins, as well as sclerotia of the same species, have been examined. The results demonstrated that the heterokaryotic condition is rare in the cultures, but it is frequent in the mycelium from sclerotia. Since it is known that the production of alkaloids is typical of the sclerotial phase in C. purpurea, it is suggested that this capacity is related to the heterokaryosis of the producing strains.     

Detection of toxigenic isolates of Aspergillus flavus and related species on coconut cream agar

A new readily-prepared medium, coconut cream agar, was developed for the detection of aflatoxin production by isolates of Aspergillus flavus and related species. Coconut cream agar, which comprised coconut cream (50%) and agar (1.5%), detected isolates of A. flavus more effectively than the synthetic media tested and was as effective as media containing desiccated coconut. Fluorescence colouring of colonies grown on coconut cream agar could be used to differentiate A. flavus from A. parasiticus and A. nomius. In addition, conidial colour of A. flavus and A. nomius was quite distinct from that of A. parasiticus.     

草莓炭疽病生防菌株MT-06的鉴定及生物学特性

对草莓炭疽病具有良好生防效果的菌株MT-06的抑菌谱、生物学特性及其分类地位进行了研究。结果表明:MT-06具有广谱抗菌性,对多种病原菌具有拮抗作用;其营养生长的最适条件是:在PDA或PSA培养基上,28-35℃,初始pH为5,24h全光照;在查氏培养基上,25-28℃,24h全光照条件下孢子产量最高。经形态观察和rDNA-ITS序列及β-tubulin序列分析,将该菌株鉴定为Talaromyces flavus(Klcker)Stolk Samson。     

Variations in UV-B tolerance and germination speed of Metarhizium anisopliae conidia produced on insects and artificial substrates

Solar ultraviolet radiation (UV-A and UV-B) is a major factor in failure of programs using the insect pathogenic fungus Metarhizium anisopliae as a biological control agent. Studies were conducted to determine if growth conditions, viz. artificial (agar media or rice grain) or natural (infected insects) substrates for conidial production affect two traits that directly influence performance of conidia after field application: tolerance to UV-B radiation and conidial germination speed. Conidia of two isolates (ARSEF 23 and ARSEF 2575) of M. anisopliae var. anisopliae produced on potato dextrose agar plus yeast extract (PDAY) or on fungus-killed larvae of two insect species, Galleria mellonella and Zophobas morio, were inactivated by exposure to UV-B radiation. Conidia of both isolates when produced on insect cadavers were significantly more sensitive to UV-B radiation than conidia produced on PDAY. Also, conidia from insect cadavers germinated slower than those from PDAY cultures. A comparison of conidia from artificial substrates showed that conidia produced on Czapek's and Emerson's YpSs agar media or rice grains had higher tolerance to UV-B radiation and germinated faster than conidia raised on PDA and PDAY. Accordingly, the growth substrate and nutritional environment in which conidia are produced influences M. anisopliae conidial UV-B tolerance and speed of germination; and manipulation of these variables could be used to obtain conidia with increased tolerance to UV-B radiation and shorter germination times.     

Divergent regulation of aflatoxin production at acidic pH by two <Emphasis Type="Italic">Aspergillus</Emphasis> strains

Production of aflatoxins (AF) by Aspergillus flavus and A. parasiticus is known to occur only at acidic pH. Although typical A. flavus isolates produced more AF as the external pH became increasingly acidic, an atypical strain from West Africa produced less. The lower AF production was not well correlated with decreases in expression of the aflatoxin pathway regulatory gene, aflR, or of two other biosynthesis genes.     

Metarhizium robertsii illuminated during mycelial growth produces conidia with increased germination speed and virulence

Light conditions during fungal growth are well known to cause several physiological adaptations in the conidia produced. In this study, conidia of the entomopathogenic fungi Metarhizium robertsii were produced on: 1) potato dextrose agar (PDA) medium in the dark; 2) PDA medium under white light (4.98 W m^?2); 3) PDA medium under blue light (4.8 W m^?2); 4) PDA medium under red light (2.8 W m^?2); and 5) minimum medium (Czapek medium without sucrose) supplemented with 3 % lactose (MML) in the dark. The conidial production, the speed of conidial germination, and the virulence to the insect Tenebrio molitor (Coleoptera: Tenebrionidae) were evaluated. Conidia produced on MML or PDA medium under white or blue light germinated faster than conidia produced on PDA medium in the dark. Conidia produced under red light germinated slower than conidia produced on PDA medium in the dark. Conidia produced on MML were the most virulent, followed by conidia produced on PDA medium under white light. The fungus grown under blue light produced more conidia than the fungus grown in the dark. The quantity of conidia produced for the fungus grown in the dark, under white, and red light was similar. The MML afforded the least conidial production. In conclusion, white light produced conidia that germinated faster and killed the insects faster; in addition, blue light afforded the highest conidial production.     

Factors affecting germination, mycoparasitism, and survival of Sporidesmium sclerotivorum.

Macroconidia of Sporidesmium sclerotivorum, a mycoparasite of Sclerotinia spp., germinated after 3 days in soil adjacent to sclerotia of S. minor and on membrane filters placed on soil containing sclerotia. Germination increased with time up to 18 days and with concentration of sclerotia. Conidia as distant as 9 mm from single sclerotia germinated. Germination of conidia was maximum on a sclerotial agar medium in the range of pH 5 to pH 7. Cultivation of S. sclerotivorum parasitically on living sclerotia proceeded optimally in moist, fine quartz sand amended with 1 to 2% (w/w) sclerotia and 0.07% (w/w) CaCO3, at 25 degrees C. Infection of sclerotia in sand reached 100% by 5 weeks. Conidia production paralled infection resulting in logarithmic increase in numbers; a maximum of 3 x 10(5) to 4 x 10(5) conidia/g was reached in 6 to 12 weeks. Viability of air-dried sand-sclerotial cultures of S. sclerotivorum was reduced after 1 and 6 days, but viability was undiminished in air-dried soil. Sporidesmium sclerotivorum survived in moist and air-dried soils stored at room temperature for 15 months.     

Description of a Distinctive Aflatoxin-Producing Strain of <Emphasis Type="Italic">Aspergillus nomius</Emphasis> that Produces Submerged Sclerotia

A new distinctive strain of Aspergillus nomius that produces the potent mycotoxins, aflatoxins, is described from pistachio, pecan, and fig orchards in California. Similar to the typical strain of A. nomius (as represented by the ex-type), the O strain produced both B and G aflatoxins but not cyclopiazonic acid, had similar conidial ornamentation, and grew poorly at 42°C. Furthermore, previous published DNA sequence supports that the new strain is very closely related to the ex-type of A. nomius. However, the O strain differs from the ex-type in several morphological characters. The ex-type was initially described as producing “indeterminate sclerotia” that appear as large (up to 3 mm long) elongated sclerotia on surfaces of media. The O strain produces only small spherical sclerotia (mean diameter <0.3 mm) submerged in the medium. In addition, the O strain has predominantly uniseriate conidial heads, whereas the typical strain of A. nomius has predominantly biseriate heads. The O strain colony color on both Czapek solution agar and Czapek yeast extract agar was more yellowish than the ex-type of A. nomius and other common aflatoxin-producing fungi. Isolates of the O strain reported here from several orchards represent the first report of A. nomius in California.     

CmPEX6, a Gene Involved in Peroxisome Biogenesis,Is Essential for Parasitism and Conidiation by the Sclerotial Parasite Coniothyrium minitans

Coniothyrium minitans is a sclerotial parasite of the plant-pathogenic fungus Sclerotinia sclerotiorum, and conidial production and parasitism are two important aspects for commercialization of this biological control agent. To understand the mechanism of conidiation and parasitism at the molecular level, we constructed a transfer DNA (tDNA) insertional library with the wild-type strain ZS-1. A conidiation-deficient mutant, ZS-1TN22803, was uncovered through screening of this library. This mutant could produce pycnidia on potato dextrose agar (PDA), but most were immature and did not bear conidia. Moreover, this mutant lost the ability to parasitize or rot the sclerotia of S. sclerotiorum. Analysis of the tDNA flanking sequences revealed that a peroxisome biogenesis factor 6 (PEX6) homolog of Saccharomyces cerevisiae, named CmPEX6, was disrupted by the tDNA insertion in this mutant. Targeted gene replacement and gene complementation tests confirmed that a null mutation of CmPEX6 was responsible for the phenotype of ZS-1TN22803. Further analysis showed that both ZS-1TN22803 and the targeted replacement mutants could not grow on PDA medium containing oleic acid, and they produced much less nitric oxide (NO) and hydrogen peroxide (H₂O₂) than wild-type strain ZS-1. The conidiation of ZS-1TN22803 was partially restored by adding acetyl-CoA or glyoxylic acid to the growth media. Our results suggest that fatty acid β-oxidation, reactive oxygen and nitrogen species, and possibly other unknown pathways in peroxisomes are involved in conidiation and parasitism by C. minitans.     

Taxonomic comparison of three different groups of aflatoxin producers and a new efficient producer of aflatoxin B1, sterigmatocystin and 3-O-methylsterigmatocystin, Aspergillus rambellii sp. nov

Accumulation of the carcinogenic mycotoxin aflatoxin B, has been reported from members of three different groups of Aspergilli (4) Aspergillus flavus, A. flavus var. parvisclerotigenus, A. parasiticus, A. toxicarius, A. nomius, A. pseudotamarii, A. zhaoqingensis, A. bombycis and from the ascomycete genus Petromyces (Aspergillus section Flavi), (2) Emericella astellata and E. venezuelensis from the ascomycete genus Emericella (Aspergillus section Nidulantes) and (3) Aspergillus ochraceoroseus from a new section proposed here: Aspergillus section Ochraceorosei. We here describe a new species, A. rambellii referable to Ochraceorosei, that accumulates very large amounts of sterigmatocystin, 3-O-methylsterigmatocystin and aflatoxin B1, but not any of the other known extrolites produced by members of Aspergillus section Flavi or Nidulantes. G type aflatoxins were only found in some of the species in Aspergillus section Flavi, while the B type aflatoxins are common in all three groups. Based on the cladistic analysis of nucleotide sequences of ITS1 and 2 and 5.8S, it appears that type G aflatoxin producers are paraphyletic and that section Ochraceorosei is a sister group to the sections Flavi, Circumdati and Cervini, with Emericella species being an outgroup to these sister groups. All aflatoxin producing members of section Flavi produce kojic acid and most species, except A. bombycis and A. pseudotamarii, produce aspergillic acid. Species in Flavi, that produce B type aflatoxins, but not G type aflatoxins, often produced cyclopiazonic acid. No strain was found which produce both G type aflatoxins and cyclopiazonic acid. It was confirmed that some strains of A. flavus var. columnaris produce aflatoxin B2, but this extrolite was not detected in the ex type strain of that variety. A. flavus var. parvisclerotigenus is raised to species level based on the specific combination of small sclerotia, profile of extrolites and rDNA sequence differences. A. zhaoqingensis is regarded as a synonym of A. nomius, while A. toxicarius resembles A. parasiticus but differs with at least three base pair differences. At least 10 Aspergillus species can be recognized which are able to biosynthesize aflatoxins, and they are placed in three very different clades.     

Positive correlation exists between glutathione S-transferase activity and aflatoxin formation in Aspergillus flavus.

The presence of glutathione (GSH) S-transferase activity, using 1-chloro-2, 4-dinitrobenzene (CDNB) as a substrate, has been established in the cytosolic fraction of the toxigenic (aflatoxin producing) and nontoxigenic strains of Aspergillus flavus. Significant differences in the GSH S-transferase activity were observed between the toxigenic and non-toxigenic strains. A positive correlation has been demonstrated for the first time between aflatoxin formation and a biochemical parameter, namely GSH S-transferase activity. The evidence in support of A. flavus GSH S-transferase induction by endogenous aflatoxins is as follows: (i) the age-related production of aflatoxin follows the same pattern as the cytosolic GSH S-transferase activity profile; (ii) significantly higher enzyme activity was associated with mycelia of a toxigenic strain grown in medium supporting high aflatoxin production (sucrose-low-salts medium) while the enzyme activity was low in medium producing less aflatoxin (glucose-ammonium nitrate medium). The GSH S-transferase activity of the non-toxigenic strain was hardly affected by a change in the medium as it produces no aflatoxins; and (iii) the toxigenic strain demonstrated significantly higher apparent Vmax. with no change in Km as compared with the non-toxigenic strain. This indicates that the enzyme induction by endogenous aflatoxins is similar to the action of phenobarbitol and other inducing drugs (Kaplowitz et al., 1975).