Study of Spanish grape mycobiota and ochratoxin A production by Isolates of Aspergillus tubingensis and other members of Aspergillus section Nigri

The native mycobiota of five grape varieties grown in Spain has been studied. Four (Bobal, Tempranillo, Garnacha, and Monastrell) were red varieties and one (Moscatel) was white. The main fungal genera isolated were Alternaria, Cladosporium, and Aspergillus. The isolation frequency of Aspergillus spp. section Nigri in contaminated samples was 82%. Ochratoxin A (OTA) production was assessed using yeast extract-sucrose broth supplemented with 5% bee pollen. Cultures of 205 isolates from this section showed that 74.2% of Aspergillus carbonarius and 14.3% of Aspergillus tubingensis isolates produced OTA at levels ranging from 1.2 to 3,530 ng/ml and from 46.4 to 111.5 ng/ml, respectively. No Aspergillus niger isolate had the ability to produce this toxin under the conditions assayed. Identification of the A. niger aggregate isolates was based on PCR amplification of 5.8S rRNA genes and its two intergenic spacers, internal transcribed spacer 1 (ITS1) and ITS2, followed by digestion with restriction endonuclease RsaI of the PCR products. The restriction patterns were compared with those from strains of A. niger CECT 2807 and A. tubingensis CECT 20393, held at the Spanish Collection of Type Cultures. DNA sequencing of the ITS1-5.8S rRNA gene-ITS2 region of the OTA-producing isolates of A. tubingensis matched 99 to 100% with the nucleotide sequence of strain A. tubingensis CBS 643.92. OTA determination was accomplished by liquid chromatography with fluorescence detection. OTA confirmation was carried out by liquid chromatography coupled to ion trap mass spectrometry. The results showed that there are significant differences with regard to the isolation frequency of ochratoxinogenic fungi in the different grape varieties. These differences were uncorrelated to berry color. The ability of A. tubingensis to produce OTA and the influence of grape variety on the occurrence of OTA-producing fungi in grapes are described in this report for the first time.     

Interpretation of mtDNA RFLP variability among <Emphasis Type="Italic">Aspergillus tubingensis</Emphasis> isolates

Aspergillus tubingensis isolates collected from distant geographic areas were earlier classified into six groups on the basis of the mtDNA RFLP variability they exhibited (mtDNA types 2a-2f). In the present work, we investigated the reason for the intraspecific mtDNA variability and we describe here how this fungus, with a relatively small mitochondrial genome, can display intraspecific polymorphism due to intron acquisition and also sporadic point mutations affecting the recognition motifs of the restriction enzymes employed in the RFLP analysis. Three different LAGLI-DADG type group I introns were identified in the cox1 gene amongst the six mtDNA RFLP types. MtDNAs of types 2b and 2d contain all of the three introns, mtDNA of type 2f carries only one, and the other mtDNA types contain two introns each. Comparative analysis showed that the first and second introns of mtDNAs of types 2b and 2d are well distributed among fungi, indicating their active horizontal transfer capacity. The third intron occurs rarely among fungi and is restricted to a limited number of fungal species, namely to A. tubingensis and the yeast Candida stellata. It is interesting that this intron is present in a small mitochondrial genome such as that of A. tubingensis and, considering its rarity, its presence amongst black Aspergillus isolates is recommended to be considered as a tool to establish taxonomical unit(s) or to track down evolutionary divergence of closely related taxonomical units.     

Fluoride-Tolerant Mutants of Aspergillus niger Show Enhanced Phosphate Solubilization Capacity

P-solubilizing microorganisms are a promising alternative for a sustainable use of P against a backdrop of depletion of high-grade rock phosphates (RPs). Nevertheless, toxic elements present in RPs, such as fluorine, can negatively affect microbial solubilization. Thus, this study aimed at selecting Aspergillus niger mutants efficient at P solubilization in the presence of fluoride (F⁻). The mutants were obtained by exposition of conidia to UV light followed by screening in a medium supplemented with Ca₃(PO₄)₂ and F⁻. The mutant FS1-555 showed the highest solubilization in the presence of F⁻, releasing approximately 70% of the P contained in Ca₃(PO₄)₂, a value 1.7 times higher than that obtained for the wild type (WT). The mutant FS1-331 showed improved ability of solubilizing fluorapatites, increasing the solubilization of Araxá, Catalão, and Patos RPs by 1.7, 1.6, and 2.5 times that of the WT, respectively. These mutants also grew better in the presence of F⁻, indicating that mutagenesis allowed the acquisition of F⁻ tolerance. Higher production of oxalic acid by FS1-331 correlated with its improved capacity for RP solubilization. This mutant represents a significant improvement and possess a high potential for application in solubilization systems with fluoride-rich phosphate sources.     

New PCR method to differentiate species in the Aspergillus niger aggregate

The DNA that encodes the 5.8S gene of the ribosomal RNA and the two intergenic spacers ITS1 and ITS2 of the two proposed type strains of the Aspergillus niger aggregate (A. niger and Aspergillus tubingensis) have been sequenced. By comparison of sequences we have found that both species could be differentiated by RsaI digestion of the PCR products of the mentioned regions. This method could be a useful tool in the identification of strains of the A. niger aggregate, especially in studies that involve a large number of isolates.     

Effect of Carbon and Nitrogen Sources on Phosphate Solubilization by a Wild-Type Strain and UV-Induced Mutants of Aspergillus tubingensis

The mechanisms of action of phosphate solubilization were studied in the wild-type strain Aspergillus tubingensis and the phenotypic mutants derived from it. The P solubilization activities of these isolates were measured in liquid media using different carbon and nitrogen sources. All the mutants showed higher P solubilization compared to the wild type. Glucose and sucrose significantly promoted P solubilization compared to fructose, lactose, galactose, and xylose. Potassium nitrate significantly increased P solubilization compared to other nitrogen sources such as ammonium sulfate, ammonium nitrate, aspargine, and tryptophan. The P solubilization activity was strongly associated with the production of organic acids, especially succinic acid and acetic acid. The enzyme activities such as acid phosphatase and phytase also increased significantly in mutants compared to the wild type. These results suggested the role of these enzymes in P solubilization apart from the organic acid exudation and H⁺ pump in A. tubingensis.     

Aflatoxin production by Aspergillus flavus isolates pathogenic to coconut insect pests

Aspergillus flavus isolated from naturally infected leaf-eating caterpillar (Opisina arenosella W.), lace bug (Stephanitis typica D.) and plant hopper (Proutista moesta Westwood), insect pests of the coconut palm, were tested for aflatoxin (AT) production by employing various media formulations. These A. flavus isolates were earlier found to be entomopathogenic in laboratory bioassays. A laboratory contaminant and four standard aflatoxigenic A. flavus isolates were also included in this study as reference strains. All A. flavus isolates were tested on seven AT detection media: coconut extract agar, coconut extract-sodium desoxycholate agar, coconut extract-ascorbic acid agar, coconut extract-Czapek Dox agar, coconut extract-milk powder agar, 10% commercial coconut milk powder agar (CCMPA) and 20% CCMPA. Only two isolates of A. flavus, originally isolated from O. arenosella and P. moesta, produced ATs. AT production was detected within 48 h of incubation and was detected continually up to 1 month. These AT-producing A. flavus isolates also produced bright yellow pigmentation in the medium. Of all the seven media used for AT detection, CCMPA (10%) was found to be the best one, followed by 20% CCMPA, for direct and rapid AT detection. AT production was not necessary for pathogenicity in the insects. This revised version was published online in July 2006 with corrections to the Cover Date.     

Comparative analysis of the complete mitochondrial genomes of Aspergillus niger mtDNA type 1a and Aspergillus tubingensis mtDNA type 2b.

To understand the differences in the organization of mitochondrial genomes of the very closely related Aspergillus niger and Aspergillus tubingensis species, we determined the complete genome sequence of the 1a mtDNA type of A. niger and 2b mtDNA type of A. tubingensis and now we provide a comparative analysis of the two mtDNAs. We found that (1) the organization (gene content and order) of the two genomes is almost identical and (2) the size difference between them is principally attributed to the different intron content of their cox1, atp9 and ndh4L genes.     

The faeA genes from Aspergillus niger and Aspergillus tubingensis encode ferulic acid esterases involved in degradation of complex cell wall polysaccharides.

We report the cloning and characterization of a gene encoding a ferulic acid esterase, faeA, from Aspergillus niger and Aspergillus tubingensis. The A. niger and A. tubingensis genes have a high degree of sequence identity and contain one conserved intron. The gene product, FAEA, was overexpressed in wild-type A. tubingensis and a protease-deficient A. niger mutant. Overexpression of both genes in wild-type A. tubingensis and an A. niger protease-deficient mutant showed that the A. tubingensis gene product is more sensitive to degradation than the equivalent gene product from A. niger. FAEA from A. niger was identical to A. niger FAE-III (C. B. Faulds and G. Williamson, Microbiology 140:779-787, 1994), as assessed by molecular mass, pH and temperature optima, pI, N-terminal sequence, and activity on methyl ferulate. The faeA gene was induced by growth on wheat arabinoxylan and sugar beet pectin, and its gene product (FAEA) released ferulic acid from wheat arabinoxylan. The rate of release was enhanced by the presence of a xylanase. FAEA also hydrolyzed smaller amounts of ferulic acid from sugar beet pectin, but the rate was hardly affected by addition of an endo-pectin lyase.     

溶磷菌对4种难溶性磷酸盐溶解能力的初步研究

以4种难溶性磷酸盐为培养基,发现供试菌株溶解这些磷酸盐的特性差异很大,真菌溶磷能力普遍比细菌要高得多。以NO3-为氮源时的溶磷量通常高于以NH4+为氮源时的溶磷量,只有2TCiF2对氟磷灰石及4TCiF6对磷酸铝的溶解能力以NH4+为氮源时较高。大多数菌株较易溶解CaP(氟磷灰石和磷矿粉),其次为AlP(AlPO4),而溶解FeP(FePO4·4H2O的能力都比较弱,只有曲霉2TCiF2具有较强的溶解FeP能力,尤其是当供给NO3-时,溶解FeP的活性比供给NH4+时大幅度提高。欧文氏菌4TCRi22和肠杆菌1TCRi15能大量地溶解氟磷灰石,而两株节杆菌对磷矿粉的溶解能力最强。供试菌株的溶磷作用可能是由于分泌的有机酸与金属离子络合或螯合作用所致,欧文氏菌和肠杆菌溶解难溶性磷过程中,非有机酸物质可能在起主要作用。     

A new black Aspergillus species, A. vadensis, is a promising host for homologous and heterologous protein production

A new species of the group of black aspergilli, Aspergillus vadensis, was analyzed for its potential as a host for homologous and heterologous protein production. Unlike the other black aspergilli, this strain does not acidify the culture medium when nitrate is the nitrogen source and only produces very low levels of extracellular proteases, mainly serine metalloproteases. The stability of A. tubingensis feruloyl esterase A (FaeA) was compared upon production in wild-type A. vadensis, A. tubingensis, and an A. niger strain in which the three main protease-encoding genes were disrupted. The production of FaeA in A. vadensis resulted in larger amounts of intact protein than production in A. tubingensis and was similar to production in an A. niger protease disruptant, confirming in vivo the low proteolytic activity of A. vadensis. The protoplast formation and transformation efficiencies of A. vadensis were much higher than those of A. niger. These characteristics make A. vadensis a very promising candidate for homologous, and possibly heterologous, protein production.     

Combined molecular and biochemical approach identifies Aspergillus japonicus and Aspergillus aculeatus as two species

We examined nine Aspergillus japonicus isolates and 10 Aspergillus aculeatus isolates by using molecular and biochemical markers, including DNA sequences of the ITS1-5.8S rRNA gene-ITS2 region, restriction fragment length polymorphisms (RFLP), and secondary-metabolite profiles. The DNA sequence of the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene could not be used to distinguish between A. japonicus and A. aculeatus but did show that these two taxa are more closely related to each other than to other species of black aspergilli. Aspergillus niger pyruvate kinase (pkiA) and pectin lyase A (pelA) and Agaricus bisporus 28S rRNA genes, which were used as probes in the RFLP analysis, revealed clear polymorphism between these two taxa. The A. niger pkiA and pelA probes placed six strains in an A. japonicus group and 12 isolates in an A. aculeatus group, which exhibited intraspecific variation when they were probed with the pelA gene. The secondary-metabolite profiles supported division of the isolates into the two species and differed from those of other black aspergilli. The strains classified as A. japonicus produced indole alkaloids and a polar metabolite, while the A. aculeatus isolates produced neoxaline, okaramins, paraherquamidelike compounds, and secalonic acid. A. aculeatus CBS 114.80 showed specific RFLP patterns for all loci examined. The secondary-metabolite profile of strain CBS 114.80 also differed from those of A. japonicus and A. aculeatus. Therefore, this strain probably represents a third taxon. This study provides unambiguous criteria for establishing the taxonomic positions of isolates of black aspergilli, which are important in relation to industrial use and legal protection of these organisms.     

Mechanisms of phosphate solubilization by fungal isolates when exposed to different P sources

The use of phosphate-solubilizing fungi is a promising biotechnological strategy in the management of phosphorus (P) fertilization, as it enables the utilization of rock phosphates (RP) or the recovery of P fixed in soil particles. The objective of our study was to evaluate fungal isolates for mechanisms of solubilization of P-bearing compounds, such as AlPO₄, FePO₄, Ca₃(PO₄)₂, Araxá RP, and Catalão RP. Four fungal isolates obtained from Brazilian soils were characterized in liquid media: Aspergillus niger FS1, Penicillium canescens FS23, Eupenicillium ludwigii FS27, and Penicillium islandicum FS30. A. niger FS1 was the only isolate able to solubilize all of the P sources, solubilizing 71, 36, 100, and 14 % of the P from AlPO₄, FePO₄, Ca₃(PO₄)₂, and RPs, respectively. Medium acidification was an effective solubilization mechanism, particularly for Ca₃(PO₄)₂. The other P sources were mainly solubilized through organic acids produced by the fungi. Oxalic acid, produced exclusively by A. niger FS1, and citric acid were decisive factors in the solubilization of AlPO₄ and FePO₄. Penicillium isolates produced more gluconic acid than A. niger FS1 in all treatments. However, this higher production did not result in higher solubilization for any of the P sources, showing that gluconic acid contributes little to the solubilization of the P sources evaluated. The higher capacity of medium acidification and the production of organic acids with stronger metal-complexation activity are characteristics that confer to A. niger FS1 a wider action on insoluble P sources. Consequently, this isolate qualifies as a promising candidate for application in the management of P fertilization.     

Ochratoxin A production and amplified fragment length polymorphism analysis of Aspergillus carbonarius, Aspergillus tubingensis, and Aspergillus niger strains isolated from grapes in Italy

Ochratoxin A is a potent nephrotoxin and a possible human carcinogen that can contaminate various agricultural products, including grapes and wine. The capabilities of species other than Aspergillus carbonarius within Aspergillus section Nigri to produce ochratoxin A from grapes are uncertain, since strain identification is based primarily on morphological traits. We used amplified fragment length polymorphisms (AFLPs) and genomic DNA sequences (rRNA, calmodulin, and beta-tubulin genes) to identify 77 black aspergilli isolated from grape berries collected in a 2-year survey in 16 vineyards throughout Italy. Four main clusters were distinguished, and they shared an AFLP similarity of <25%. Twenty-two of 23 strains of A. carbonarius produced ochratoxin A (6 to 7,500 microg/liter), 5 of 20 strains of A. tubingensis produced ochratoxin A (4 to 130 microg/liter), 3 of 15 strains of A. niger produced ochratoxin A (250 to 360 microg/liter), and none of the 19 strains of Aspergillus "uniseriate" produced ochratoxin A above the level of detection (4 microg/liter). These findings indicate that A. tubingensis is able to produce ochratoxin and that, together with A. carbonarius and A. niger, it may be responsible for the ochratoxin contamination of wine in Italy.     

Progesterone transformation as a diagnostic feature in the classification of the Aspergillus niger group

Fifty-five isolates of nine species and two species varieties (5 isolates each) of the Aspergillus niger group were tested for progesterone transformation. Generally, all isolates of the different species transformed progesterone to 21-hydroxyprogesterone. In addition, the isolates of seven species have the capacity to hydroxylate progesterone to 11α-hydroxyprogesterone. A systematic variation could be observed between the tested species of the Aspergillus niger group with respect to the transformation reactions of progesterone. Comparative biotransformation results showed that essential differences exist between the species of this group, where the different isolates of individual species always produce one or more identical products. This biochemical differentiation may supplement the morphological criteria used in the classification of this group of fungi.     

Aspergillus niger contains the cryptic phylogenetic species A. awamori

Aspergillus section Nigri is an important group of species for food and medical mycology, and biotechnology. The Aspergillus niger 'aggregate' represents its most complicated taxonomic subgroup containing eight morphologically indistinguishable taxa: A. niger, Aspergillus tubingensis, Aspergillus acidus, Aspergillus brasiliensis, Aspergillus costaricaensis, Aspergillus lacticoffeatus, Aspergillus piperis, and Aspergillus vadensis. Aspergillus awamori, first described by Nakazawa, has been compared taxonomically with other black aspergilli and recently it has been treated as a synonym of A. niger. Phylogenetic analyses of sequences generated from portions of three genes coding for the proteins β-tubulin (benA), calmodulin (CaM), and the translation elongation factor-1 alpha (TEF-1α) of a population of A. niger strains isolated from grapes in Europe revealed the presence of a cryptic phylogenetic species within this population, A. awamori. Morphological, physiological, ecological and chemical data overlap occurred between A. niger and the cryptic A. awamori, however the splitting of these two species was also supported by AFLP analysis of the full genome. Isolates in both phylospecies can produce the mycotoxins ochratoxin A and fumonisin B?, and they also share the production of pyranonigrin A, tensidol B, funalenone, malformins, and naphtho-γ-pyrones. In addition, sequence analysis of four putative A. awamori strains from Japan, used in the koji industrial fermentation, revealed that none of these strains belong to the A. awamori phylospecies.     

Inhibition of species of the Aspergillus section Nigri and ochratoxin a production in grapes by fusapyrone

Fusapyrone (FP), an antifungal natural compound, was tested against the three main ochratoxigenic species of the Aspergillus section Nigri. The MICs at 24 h were 6.0, 11.6, and 9.9 mug/ml for Aspergillus carbonarius, Aspergillus tubingensis, and Aspergillus niger, respectively. Strong inhibition of growth and morphological changes were still observed at half the MIC after 7 days. The application of a 100 mug/ml FP solution in a laboratory assay on artificially inoculated grapes resulted in a significant reduction (up to 6 orders of magnitude) of A. carbonarius CFU counts. Dramatic reductions of the ochratoxin A (OTA) content, compared to the content of the positive control (average amount of OTA, 112.5 ng/g of grape; three experiments), were obtained with the application of either 100 or 50 mug/ml of FP (0.6 or 5.1 ng/g of grape, respectively).     

Comparison of cultural and analytical methods for determination of aflatoxin production by Mississippi Delta Aspergillus isolates

This study compared cultural and analytical methods for detecting aflatoxin production by Aspergillus species. Aspergillus isolates were obtained from various Mississippi Delta crops (corn, peanut, rice, cotton) and soils. Most of the isolates (99%) were A. flavus and the remainder comprised A. parasiticus and A. nomius. The following three cultural methods were evaluated on potato dextrose agar: fluorescence (FL) on beta-cyclodextrin-containing media (CD), yellow pigment (YP) formation in mycelium and medium, and color change after ammonium hydroxide vapor exposure (AV). Aflatoxins in culture extracts were confirmed by thin-layer chromatography (TLC) and quantified by enzyme-linked immunosorbent assay (ELISA). Of the 517 isolates, 314 produced greater than 20 ng/g of total aflatoxin based on ELISA, and 180 produced greater than 10 000 ng/g of aflatoxin in the medium. Almost all the toxigenic isolates (97%) were confirmed by TLC as producers. Of the toxigenic isolates, as determined by ELISA, 93%, 73%, and 70% gave positive FL, YP, and AV responses, respectively. Of the 203 isolates producing less than 20 ng/g of aflatoxin, 20%, 6%, and 0% of respective FL, YP, and AV methods gave false-positive responses. The 9% false-positive results from TLC fall within this range. This study showed good agreement among all tested cultural methods. However, these cultural techniques did not detect aflatoxin in all cultures that were found to produce aflatoxins by ELISA, LC/MS, and TLC. The best results were obtained when the AV color change and CD fluorescence methods were used together, yielding an overall success rate comparable to TLC but without the need for chemical extraction and the time and expense of TLC.     

Discrimination of Aspergillus niger and other Aspergillus species belonging to section Nigri by PCR assays

Aspergillus species included in section Nigri are common in plant products and processed food, such as grapes, cereals, coffee and derivatives, particularly in warm and tropical climates. Two of these species, A. carbonarius and A. niger, are known to produce ochratoxin A (OTA), a potent nephrotoxin and carcinogenic to human (group 2B). Recognition of the several species of this section is difficult and requires considerable expertise using conventional methods based on morphological features. In this work we describe rapid, sensitive and robust assays based on the PCR technique to discriminate the main species included in section Nigri: A. japonicus, A. heteromorphus, A. ellipticus and the two morphologically indistinguishable species of the A. niger aggregate: A. niger and A. tubingensis. The species-specific primers have been designed on the basis of ITS (internal transcribed spacers of rDNA units) sequence comparisons obtained from several Aspergillus strains and have been tested in a number of strains from different origins and hosts. These PCR assays, based on multi-copy sequences, are highly sensitive and specific and represent a good tool for an early detection of OTA-producing Aspergillus species in order to prevent OTA from entering the food chain.     

Phosphorus transformations from rock phosphates in acid soils and measures for increasing their efficiency for growing rice (Oryza sativa L.)

Summary Laboratory incubation experiments showed that addition of rock phosphate to P-deficient acid red and laterite soils resulted in an increase in Al–P or/and Fe–P, with a consequent decrease in Ca–P during 15 days, of moist aerobic incubation. The transformations of P from Gafsa, Jordan, North Carolina and Florida rock phosphates were more than those from Tennessee, Missouri and Udaipur. Studies with North Carolina, Gafsa, and Udaipur rock phosphates showed that application of the former two to moist aerobic P-deficient acid soils 2 weeks prior to flooding and transplanting rice gave higher content of Al–P, Fe–P and Bray-P, compared to when these were applied at flooding. The grain yields obtained with the former two treatments were also at par with that obtained with the addition of superphosphate at comparable rate (100 ppm) of P application, compared to when the, rock phosphates were applied at flooding, where the grain yields were lower than the superphosphate treatment, indicating that some of these rock phosphates could be made as efficient as superphosphate for growing rice on acid soils by their application to moist aerobic soil, 2–3 weeks prior to flooding and transplanting rice and thus conserve some amount of sulphur required for the manufacture of water soluble phosphates.     

Rock phosphate solubilization by psychrotolerant Pseudomonas spp. and their effect on lentil growth and nutrient uptake under polyhouse conditions

Psychrotolerant Pseudomonas isolates (RT5RP2 and RT6RP) isolated from the rhizoplane of wild grass at 3,100 and 3,800 m above mean sea level, respectively, from Rudraprayag district of Uttarakhand (India), were found to solubilize Udaipur rock phosphate (URP). Both isolates grew at temperatures ranging from 4 to 30 °C. Kinetics of phosphate solubilization by the bacterial strains showed a nonlinear regression of the rate of P solubilization, which fitted best in the power model, and showed a declining trend across three different temperatures. Under pot culture conditions, bacterization of lentil seeds (cv. VL Masoor 507) with the psychrotolerant Pseudomonas strains when combined with URP as a sole source of phosphorus resulting in significant enhancement in P uptake of the plants, compared to the application of rock phosphate alone.