Comparison of major biocontrol strains of non-aflatoxigenic Aspergillus flavus for the reduction of aflatoxins and cyclopiazonic acid in maize

Biological control of toxigenic Aspergillus flavus in maize through competitive displacement by non-aflatoxigenic strains was evaluated in a series of field studies. Four sets of experiments were conducted between 2007 and 2009 to assess the competitiveness of non-aflatoxigenic strains when challenged against toxigenic strains using a pin-bar inoculation technique. In three sets of experiments the non-aflatoxigenic strain K49 effectively displaced toxigenic strains at various concentrations or combinations. The fourth study compared the relative competitiveness of three non-aflatoxigenic strains (K49, NRRL 21882 from Afla-Guard?, and AF36) when challenged on maize against two aflatoxin- and cyclopiazonic acid (CPA)-producing strains (K54 and F3W4). These studies indicate that K49 and NRRL 21882 are superior to AF36 in reducing total aflatoxin contamination. Neither K49 nor NRRL 21882 produce CPA and when challenged with K54 and F3W4, CPA and aflatoxins were reduced by 84-97% and 83-98%, respectively. In contrast, AF36 reduced aflatoxins by 20% with F3W4 and 93% with K54 and showed no reduction in CPA with F3W4 and only a 62% reduction in CPA with K54. Because AF36 produces CPA, high levels of CPA accumulate when maize is inoculated with AF36 alone or in combination with F3W4 or K54. These results indicate that K49 may be equally effective as NRRL 21882 in reducing both aflatoxins and CPA in maize.     

Variability of aflatoxin and cyclopiazonic acid production by Aspergillus section flavi from different substrates in Argentina

Aspergillus section flavi strains isolated from peanuts, wheat and soybean grown in Argentina were screened for aflatoxins (type B and G) and cyclopiazonic acid (CPA) production. Aspergillus flavus was the predominant species in all substrates, although there was almost the same proportion of A. flavus and Aspergillus parasiticus in peanuts. Aspergillus nomius was not found. Incidence of aflatoxigenic A. flavus strains was higher in peanuts (69%) than in wheat (13%) or soybeans (5%) while the ratio of CPA producers A. flavus isolated from all substrates was very high (94% in peanuts, 93% in wheat and 73% in soybeans). Isolates of A. flavus able to produce simultaneously aflatoxins type B and CPA were detected in all substrates, suggesting the possibility of co-occurrence of these toxins. Almost all isolates of A. parasiticus resulted aflatoxins (type B and G) producers but did not produce CPA. Five of sixty-seven strains isolated from peanuts showed an unusual pattern of mycotoxin production (aflatoxins type B and G simultaneously with CPA). These strains also produced numerous small sclerotia like S strains of A. flavus detected in cottonseed in Arizona and in soils of Thailand and West Africa. The atypical strains are not widely distributed in Argentina and were found uniquely in peanuts.     

Comparison of major biocontrol strains of non-aflatoxigenic Aspergillus flavus for the reduction of aflatoxins and cyclopiazonic acid in maize

Biological control of toxigenic Aspergillus flavus in maize through competitive displacement by non-aflatoxigenic strains was evaluated in a series of field studies. Four sets of experiments were conducted between 2007 and 2009 to assess the competitiveness of non-aflatoxigenic strains when challenged against toxigenic strains using a pin-bar inoculation technique. In three sets of experiments the non-aflatoxigenic strain K49 effectively displaced toxigenic strains at various concentrations or combinations. The fourth study compared the relative competitiveness of three non-aflatoxigenic strains (K49, NRRL 21882 from Afla-Guard®, and AF36) when challenged on maize against two aflatoxin- and cyclopiazonic acid (CPA)-producing strains (K54 and F3W4). These studies indicate that K49 and NRRL 21882 are superior to AF36 in reducing total aflatoxin contamination. Neither K49 nor NRRL 21882 produce CPA and when challenged with K54 and F3W4, CPA and aflatoxins were reduced by 84–97% and 83–98%, respectively. In contrast, AF36 reduced aflatoxins by 20% with F3W4 and 93% with K54 and showed no reduction in CPA with F3W4 and only a 62% reduction in CPA with K54. Because AF36 produces CPA, high levels of CPA accumulate when maize is inoculated with AF36 alone or in combination with F3W4 or K54. These results indicate that K49 may be equally effective as NRRL 21882 in reducing both aflatoxins and CPA in maize.     

Natural and in vitro coproduction of cyclopiazonic acid and aflatoxins

Eighty samples of animal feeds of different origins were screened for the natural co-occurrence of cyclopiazonic acid (CPA) and aflatoxins in Portugal. Forty-five strains of Aspergillus flavus were collected from those samples and studied for their ability to produce these mycotoxins, in vitro. CPA was detected by thin-layer chromatography using Erhlich's reagent for confirmation. Aflatoxins were determined by high-pressure liquid chromatography with postcolumn iodination. Only 5 of the 80 samples (6.2%) were naturally contaminated with cyclopiazonic acid (0.16 mg/kg) and 36 (45.0%) with aflatoxin B1 (AFB1) (from 0.001 to 0.016 mg/kg). An in vitro study of the 45 strains of A. flavus was performed in cracked corn at 25 degrees C (water activity, a(w) = 0.96), incubated for 21 days to CPA production. For in vitro production of aflatoxins, the same substrate was incubated at 28 degrees C for 14 days. Nineteen of the strains (42.2%) produced CPA (ranging from 0.5 to 1.45 mg of CPA/kg) and 23 of them (51.1%) produced AFB1 (from 0.001 to 0.844 mg/kg). Only 10 isolates (22.2%) produced both CPA and AFB1 (0.05 to 0.10 mg/kg and 0.001 to 0.230 mg/kg, respectively). Thirteen strains did not produce either CPA nor AFB1.     

Cladal relatedness among Aspergillus oryzae isolates and Aspergillus flavus S and L morphotype isolates

Aspergillus flavus is the main etiological agent for aflatoxin contamination of crops. Its close relative, A. oryzae, does not produce aflatoxins and has been widely used to produce fermented foods. We compared the phylogeny of A. oryzae isolates and L- and S-type sclerotial isolates of A. flavus using single nucleotide polymorphisms in the omtA gene in the aflatoxin biosynthesis gene cluster and deletions in and distal to the norB-cypA intergenic region as phylogenetic signals. Aflatoxin-producing ability and sclerotial size also were weighted in the analysis. Like A. flavus, the A. oryzae isolates form a polyphyletic assemblage. A. oryzae isolates in one clade strikingly resemble an A. flavus subgroup of atoxigenic L-type isolates. All toxigenic S-type isolates closely resemble another subgroup of atoxigenic L-type isolates. Because atoxigenic S-type isolates are extremely rare, we hypothesize that loss of aflatoxin production in S-type isolates may occur concomitantly with a change to L-type sclerotia. All toxigenic L-type isolates, unlike A. oryzae, have a 1.0 kb deletion in the norB-cypA region. Although A. oryzae isolates, like S-type, have a 1.5 kb deletion in the norB-cypA region, none were cladally related to S-type A. flavus isolates. Our results show that A. flavus populations are genetically diverse. A. oryzae isolates may descend from certain atoxigenic L-type A. flavus isolates.     

Identification of species in Aspergillus section Flavi based on sequencing of the mitochondrial cytochrome b gene.

The partial sequences of the mitochondrial (mt) cytochrome b gene (402 bp) were determined for species of Aspergillus section Flavi. On the basis of identities of DNA sequences, 77 strains were divided into seven DNA types, from D-1 to D-7. The type strains of A. sojae, A. parasiticus, A. flavus and A. oryzae together, A. tamarii, and A. nomius were placed in DNA types D-1. D-2, D-4, D-5 and D-7, respectively. These species could be differentiated from each other. Furthermore, two other DNA types, D-3 and D-6 were found. DNA type D-3 was closely related to A. parasiticus (D-2) and included one strain that deposited as A. flatus var. flavus and produced aflatoxins B and G. DNA types D-6 included one strain named A. flavus and closely related to A. tamarii. The observations of conidial wall texture by SEM (Scanning Electron Microscopy) supported the relationships derived from the cytochrome b gene. The production of aflatoxins was also examined. Using the DNA sequence of cytochrome b gene, several strains were reidentified. The derived amino acids sequences were all the same in the studied strains. The mt cytochrome b gene is useful and reliable in distinguishing and identifying the species in Aspergillus section Flavi.     

Influence of UV radiation and nitrosamines on the induction of mycotoxins synthesis by nontoxigenic moulds isolated from feed samples

The effects of UV radiation and nitrosamines on the induction of mycotoxin biosynthesis by some nontoxigenic moulds isolated from feed samples collected from Egypt and Poland was investigated. Nontoxigenic strains of Aspergillus flavus P-63, A. niger EN-200 and A. ochraceus P-157 synthesized mycotoxins (aflatoxins and ochratoxin, A) after exposure to near UV radiation for 120-210 min. Nitrosamines (DMNA and DENA) at 30 up to 1000 ppm induced the synthesis of aflatoxins by nontoxigenic species of A. flavus ES-255 and P-63 and A. niger EN 200. Near-UV radiation and nitrosamines had no influence on the induction of mycotoxin synthesis by Penicillium and Fusarium isolates. All nontoxigenic strains of Aspergilli which synthesized aflatoxins in the presence of 1000 ppm nitrosamines, also synthesized continuously aflatoxins during the next fifteen generations. Near-UV radiation and nitrosamines had a mutagenic effect on the induction of mycotoxins synthesis by nontoxigenic moulds.     

Identification and toxigenic potential of the industrially important fungi, Aspergillus oryzae and Aspergillus sojae

Mold strains belonging to the species Aspergillus oryzae and Aspergillus sojae are highly valued as koji molds in the traditional preparation of fermented foods, such as miso, sake, and shoyu, and as protein production hosts in modern industrial processes. A. oryzae and A. sojae are relatives of the wild molds Aspergillus flavus and Aspergillus parasiticus. All four species are classified to the A. flavus group. Strains of the A. flavus group are characterized by a high degree of morphological similarity. Koji mold species are generally perceived of as being nontoxigenic, whereas wild molds are associated with the carcinogenic aflatoxins. Thus, reliable identification of individual strains is very important for application purposes. This review considers the pheno- and genotypic markers used in the classification of A. flavus group strains and specifically in the identification of A. oryzae and A. sojae strains. Separation of A. oryzae and A. sojae from A. flavus and A. parasiticus, respectively, is inconsistent, and both morphologic and molecular evidence support conspecificity. The high degree of identity is reflected by the divergent identification of reference cultures maintained in culture collections. As close relatives of aflatoxin-producing wild molds, koji molds possess an aflatoxin gene homolog cluster. Some strains identified as A. oryzae and A. sojae have been implicated in aflatoxin production. Identification of a strain as A. oryzae or A. sojae is no guarantee of its inability to produce aflatoxins or other toxic metabolites. Toxigenic potential must be determined specifically for individual strains. The species taxa, A. oryzae and A. sojae, are currently conserved by societal issues.     

Mycoflora and toxigenic Aspergillus flavus in Spanish dry-cured ham

Sixty-five dry-salted hams were analysed. Aspergillus and Penicillium were the dominant genera. In general, the mould flora was dominated by Aspergillus spp. and primarily A. glaucus, A. fumigatus, A. niger and A. flavus. A flavus was found in 16 hams and 9 out of 16 strains examined produced aflatoxins 'in vitro'. Surface samples of dry-salted hams showed growth of inoculated A. parasiticus NRRL-2999 strains and production of aflatoxins in low levels at 25 and 30 degrees C. It is concluded that the presence of toxigenic strains in Spanish dry-salted ham does not constitute a health risk.     

Nonaflatoxigenic Aspergillus flavus TX9-8 competitively prevents aflatoxin accumulation by A. flavus isolates of large and small sclerotial morphotypes

Aflatoxins are a family of highly toxic and carcinogenic toxins produced by several Aspergillus species. Aflatoxin contamination of agricultural commodities both pre- and postharvest is a serious food safety issue and a significant economic concern. Using nonaflatoxigenic A. flavus isolates to competitively exclude toxigenic A. flavus isolates in agricultural fields has become an adopted approach to reduce aflatoxin contamination. From screening subgroups of nonaflatoxigenic A. flavus, we identified an A. flavus isolate, TX9-8, which competed well with three A. flavus isolates producing low, intermediate, and high levels of aflatoxins, respectively. TX9-8 has a defective polyketide synthase gene (pksA), which is necessary for aflatoxin biosynthesis. Co-inoculating TX9-8 at the same time with large sclerotial (L strain) A. flavus isolates at a ratio of 1:1 or 1:10 (TX9-8:toxigenic) prevented aflatoxin accumulation. The intervention of TX9-8 on small sclerotial (S strain) A. flavus isolates varied and depended on isolate and ratio of co-inoculation. At a ratio of 1:1 TX9-8 prevented aflatoxin accumulation by A. flavus CA28 and reduced aflatoxin accumulation 10-fold by A. flavus CA43. No decrease in aflatoxin accumulation was apparent when TX9-8 was inoculated 24 h after toxigenic L- or S strain A. flavus isolates started growing. The competitive effect likely is due to TX9-8 outgrowing toxigenic A. flavus isolates.     

Analysis of population structure of Aspergillus flavus from peanut based on vegetative compatibility, geographic origin, mycotoxin and sclerotia production

Isolates of Aspergillus flavus obtained from a new growing peanut region in Argentina (Formosa province) were examined for aflatoxin types B and G and cyclopiazonic acid (CPA) production. Sclerotia diameters and the number of sclerotia produced per square centimetre were also determined for each isolate. They were tested by vegetative compatibility group analysis to investigate their genetic relatedness and correlate the results with vegetative compatibility groups previously described from the major peanut-growing area (Córdoba province) in our country. Two isolates were considered atypical because they simultaneously produce aflatoxins B and G and CPA. A. flavus population from Formosa province was very diverse genetically. Vegetative compatibility groups (VCGs) formed by typical isolations of A. flavus were different among agroecological sites. Formosa isolates could not be grouped to any of the Córdoba VCGs, while that one of the VCGs that contain atypical isolates included strains from the two geographical regions. Each VCG included isolates of the same mycotoxin and sclerotia production pattern. The two regions analysed have different climatic conditions, soil type, crop sequence history and also are in different latitude. These parameters may reflect different geographic adaptation between isolates from both sites.     

Fungi, aflatoxins, and cyclopiazonic acid associated with peanut retailing in Botswana

Peanuts are important food commodities, but they are susceptible to fungal infestation and mycotoxin contamination. Raw peanuts were purchased from retail outlets in Botswana and examined for fungi and mycotoxin (aflatoxins and cyclopiazonic acid) contamination. Zygomycetes were the most common fungi isolated; they accounted for 41% of all the isolates and were found on 98% of the peanut samples. Among the Zygomycetes, Absidia corymbifera and Rhizopus stolonifer were the most common. Aspergillus spp. accounted for 35% of all the isolates, with Aspergillus niger being the most prevalent (20.4%). Aspergillus flavus/parasiticus were also present and accounted for 8.5% of all the isolates, with A. flavus accounting for the majority of the A. flavus/parasiticus identified. Of the 32 isolates of A. flavus screened for mycotoxin production, 11 did not produce detectable aflatoxins, 8 produced only aflatoxins B1 and B2, and 13 produced all four aflatoxins (B1, B2, G1, and G2) in varying amounts. Only 6 of the A. flavus isolates produced cyclopiazonic acid at concentrations ranging from 1 to 55 microg/kg. The one A. parasiticus isolate screened also produced all the four aflatoxins (1,200 microg/kg) but did not produce cyclopiazonic acid. When the raw peanut samples (n = 120) were analyzed for total aflatoxins, 78% contained aflatoxins at concentrations ranging from 12 to 329 microg/kg. Many of the samples (49%) contained total aflatoxins at concentrations above the 20 microg/kg limit set by the World Health Organization. Only 21% (n = 83) of the samples contained cyclopiazonic acid with concentrations ranging from 1 to 10 microg/kg. The results show that mycotoxins and toxigenic fungi are common contaminants of peanuts sold at retail in Botswana.     

Mycological survey for potential aflatoxin and ochratoxin producers and their toxicological properties in harvested Brazilian black pepper.

A mycological survey was carried out on 115 samples of whole dried black pepper seeds, from two main production regions of Brazil (Pará and Espírito Santo). A high incidence of contamination was verified in both regions when 99.1% of the samples showed filamentous fungi contamination. A total of 497 species of nine different genera were isolated (Aspergillus, Eurotium, Rhizopus, Penicillium, Curvularia, Cladosporium, Absidia, Emericella and Paecilomyces). The genus Aspergillus was the predominant (53.5%) followed by species from the Eurotium genus (24.5%). Eurotium chevalieri (16.4%) was the most predominant species followed by A. flavus (14.6%) present on 55 samples of black pepper (47.8%) analysed. Twenty-five samples (21.7%) were contaminated with aflatoxigenic strains of A. flavus and A. parasiticus. In relation to the types of aflatoxins produced by mycotoxigenic strains, it was observed that 25 strains (44.6%) of 56 isolated of A. flavus produced aflatoxins. From 12 samples, A. ochraceus species were isolated in low frequency (3.5%). Two strains of A. ochraceus from 16 isolated were producers of ochratoxin A. With respect to the aflatoxins and ochratoxin A natural contamination, none of the samples presented detectable levels of these mycotoxins using thin-layer chromatographic analysis.     

PCR detection of aflatoxin producing fungi and its limitations

Unlike bacterial toxins that are primarily peptides and are therefore encoded by a single gene, fungal toxins such as the aflatoxins are multi-ring structures and therefore require a sequence of structural genes for their biological synthesis. There is therefore no specific PCR for any one of the four biologically produced aflatoxins. Unfortunately, the structural genes presently in use for PCR detection of aflatoxin producing fungi are also involved in the synthesis of other fungal toxins such as sterigmatocystin by Aspergillus versicolor and Aspergillus nidulans and therefore lack absolute specificity for aflatoxin producing fungi (Table?1). In addition, the genomic presence of several structural genes involved in aflatoxin biosynthesis does not guarantee the production of aflatoxin by all isolates of Aspergillus flavus and Aspergillus parasiticus. The most widely used DNA target regions for discriminating Aspergillus species are those of the rDNA complex, mainly the internal transcribed spacer regions 1 and 2 (ITS1 and ITS2) and the variable regions in the 5'-end of the 28S rRNA gene. Since these sequence regions are unrelated to the structural genes involved in aflatoxin biosynthesis there successful amplification can be used for species identification but do not confirm aflatoxin production. This review therefore presents the various approaches and limitations in the use of the PCR in attempting to detect aflatoxin producing fungi.     

Fungal population and distribution of aflatoxigenic fungi in commercial almond powder products

The fungal population and distribution of aflatoxin-producing fungi in 30 samples of imported almond powder products purchased from retail markets were examined in this study. Total counts of fungi ranged from under 1.0 x 10 colony-forming units (CFU)/g to 8.5 x 10(3) CFU/g as determined with the dilution plating technique. The predominant fungi in the mould-contaminated almond samples were Aspergillus niger, A. flavus and the related species, Penicillium, Cladosporium and Rhizopus. Aflatoxin-producing ability in the isolates of A. flavus and related fungi were tested by thin layer chromatography using 2% yeast extract and 15% sucrose broth culture. Four different aflatoxigenic fungi were detected in the isolates; aflatoxins B1 and B2 were produced by some strains of A. flavus and A. parvisclerotigenus, and aflatoxins B1, B2, G1, G2 were produced by all tested strains of A. parasiticus and A. nomius. Identification of the strains was based on morphological and metabolic characters.     

Polyphasic characterization of Aspergillus section Flavi isolated from animal feeds in Algeria

In Algeria, little information is available on the population structure of Aspergillus section Flavi in raw materials and resultant animal feeds. A total of 172 isolates belonging to Aspergillus section Flavi were recovered from 57 animal feeds and identified on the basis of macro and micro-morphological characters, mycotoxin production and genetic relatedness. For the molecular analysis, sequencing of the calmodulin gene (CaM) and the internal transcribed spacer (ITS) regions were performed for representative isolates. Four distinct morphotypes were distinguished: Aspergillus flavus (78.5%), Aspergillus tamarii (19.2%), Aspergillus parasiticus (1.7%), and Aspergillus alliaceus (0.6%). All A. flavus isolates were of the L type and no correlation between sclerotia production and aflatoxigenicity was observed. Our results showed that 68% of the A. flavus strains produced aflatoxins B (AFB), and 72.7% were cyclopiazonic acid (CPA) producers. The three isolates of A. parasiticus were able to produce AFB and aflatoxins G but not CPA whereas, all the strains of A. tamarii produced only CPA. The obtained results revealed the presence of different species of Aspergillus section Flavi, among which were aflatoxin producers. This study provides evidence useful for considerations in aflatoxin control strategies.     

微生物对黄曲霉毒素的抑制机制研究进展

部分黄曲霉菌会对农作物及产品造成严重污染.受相关基因的影响,黄曲霉会产生一种对人类健康与畜禽养殖构成重大威胁的真菌毒素,即黄曲霉毒素,在田间、储藏期或运输过程中黄曲霉毒素污染均可能发生.防止黄曲霉毒素进入食物链的策略有很多,其中采用生物法防治黄曲霉污染越来越引起研究者的重视,也逐渐被人们所接受.利用微生物对黄曲霉及毒素...     

高大平房仓稻谷粮层霉菌量与优势霉菌的差异性研究

采用传统培养法对湖南与湖北两省粮库中的稻谷进行研究,对高大平房仓粮仓上中下三层的稻谷霉菌量及优势霉菌进行研究。研究结果表明:湖南省储藏一年稻谷与新入库稻谷中层霉菌数分别为6.4×10~3 CFU/g、1.3×10~3 CFU/g,相比上、下层,中层最多;湖北省储藏一年稻谷下层霉菌数为1.4×10~4 CFU/g,相比上、中层,下层最多,湖北省新入库稻谷上层霉菌数为1.4×10~4 CFU/g,相比中、下层,上层最多。通过传统的菌落培养及菌丝、孢子观察,初步判断上中下三层的优势霉菌,并结合分子生物学的方法对其ITS序列进行分析,通过PCR扩增,将扩增出来的基因序列,在GenBank进行BLAST,最终鉴定优势菌株为黄曲霉、白曲霉、聚多曲霉、内生真菌、黑曲霉。     

Characterization of filamentous fungi isolated from Moroccan olive and olive cake: toxinogenic potential of Aspergillus strains

During the 2003 and 2004 olive oil production campaigns in Morocco, 136 samples from spoiled olive and olive cake were analyzed and 285 strains were isolated in pure culture. Strains included 167 mesophilic strains belonging to ten genera: Penicillium, Aspergillus, Geotrichum, Mucor, Rhizopus, Trichoderma, Alternaria, Acremonium, Humicola, Ulocladium as well as 118 thermophilic strains isolated in 2003 and 2004, mainly belonging to six species: Aspergillus fumigatus, Paecilomyces variotii, Mucor pusillus, Thermomyces lanuginosus, Humicola grisea, and Thermoascus aurantiacus. Penicillium and Aspergillus, respectively, 32.3 and 26.9% of total isolates represented the majority of mesophilic fungi isolated. When considering total strains (including thermotolerant strains) Aspergillus were the predominant strains isolated; follow-up studies on mycotoxins therefore focused primarily on aflatoxins (AFs) and ochratoxin A (OTA) from the latter strains. All isolated Aspergillus flavus strains (9) and Aspergillus niger strains (36) were studied in order to evaluate their capacity to produce AFs and OTA, respectively, when grown on starch-based culture media. Seven of the nine tested A. flavus strains isolated from olive and olive cake produced AF B1 at concentrations between 48 and 95 microg/kg of dry rice weight. As for the A. niger strains, 27 of the 36 strains produced OTA.