Control of postharvest diseases caused by Penicillium spp. with myrtle leaf phenolic extracts: in vitro and in vivo study on mandarin fruit during storage.

BACKGROUND: In the postharvest handling of horticultural commodities, plant extracts with fungicidal activity are a valid alternative to synthetic fungicides. The fungicidal activity of myrtle leaf extracts from eight cultivars was studied in vitro against Penicillium digitatum, Penicillium italicum, and Penicillium expansum and on artificially inoculated mandarins with green and blue molds during storage for 12 days at 20 °C and 90% RH. RESULTS: Hydroxybenzoic acids, hydrolysable tannins, and flavonols were identified by high-performance liquid chromatography (HPLC). Despite sharing the same phenolic profile, extracts of eight myrtle cultivars significantly differed in the concentration of phenolics. Hydrolysable tannins are the principal subclass representing nearly 44.9% of the total polyphenols, whereas myricitrin was the most abundant flavonol in all cultivars. Myrtle extracts strongly inhibited conidial germination of the pathogens tested, although the greatest efficacy was observed against P. digitatum. At a concentration of 20 g L-1 , all the extracts completely inhibited fungi growth; only 'Angela', 'Tonina' and 'Grazia' extracts were effective at lower concentrations (15 g L-1 ). On inoculated fruit, myrtle extracts significantly controlled rot development. As a preventive treatment, 'Ilaria' and 'Maria Rita' extracts significantly reduced the rate of fruit with green mold decay lesions. When applied as a curative treatment, all the exacts decreased the incidence of decay. Against P. italicum, all the extracts applied as preventive treatments controlled decay effectively, while as curative treatment some of the extracts were not effective. All the extracts reduced the size of the infected areas. CONCLUSION: The results propose myrtle extracts as a possible natural alternative to synthetic fungicides. © 2021 Society of Chemical Industry.

Microbiology of secondary infections in Buruli ulcer lesions; implications for therapeutic interventions.

BACKGROUND: Buruli ulcer (BU) is a skin disease caused by Mycobacterium ulcerans and is the second most common mycobacterial disease after tuberculosis in Ghana and Côte d'Ivoire. M. ulcerans produces mycolactone, an immunosuppressant macrolide toxin, responsible for the characteristic painless nature of the infection. Secondary infection of ulcers before, during and after treatment has been associated with delayed wound healing and resistance to streptomycin and rifampicin. However, not much is known of the bacteria causing these infections as well as antimicrobial drugs for treating the secondary microorganism. This study sought to identify secondary microbial infections in BU lesions and to determine their levels of antibiotic resistance due to the prolonged antibiotic therapy required for Buruli ulcer. RESULTS: Swabs from fifty-one suspected BU cases were sampled in the Amansie Central District from St. Peters Hospital (Jacobu) and through an active case surveillance. Forty of the samples were M. ulcerans (BU) positive. Secondary bacteria were identified in all sampled lesions (N = 51). The predominant bacteria identified in both BU and Non-BU groups were Staphylococci spp and Bacilli spp. The most diverse secondary bacteria were detected among BU patients who were not yet on antibiotic treatment. Fungal species identified were Candida spp, Penicillium spp and Trichodema spp. Selected secondary bacteria isolates were all susceptible to clarithromycin and amikacin among both BU and Non-BU patients. Majority, however, had high resistance to streptomycin. CONCLUSIONS: Microorganisms other than M. ulcerans colonize and proliferate on BU lesions. Secondary microorganisms of BU wounds were mainly Staphylococcus spp, Bacillus spp and Pseudomonas spp. These secondary microorganisms were less predominant in BU patients under treatment compared to those without treatment. The delay in healing that are experienced by some BU patients could be as a result of these bacteria and fungi colonizing and proliferating in BU lesions. Clarithromycin and amikacin are likely suitable drugs for clearance of secondary infection of Buruli ulcer.

Capability of Penicillium oxalicum y2 to release phosphate from different insoluble phosphorus sources and soil.

Due to insufficient amount of soluble phosphate and poor persistence of traditional chemical phosphate fertilizers in agricultural soils, the eco-friendly and sustainable phosphorus sources for crops are urgently required. The efficient phosphate-releasing fungal strain designated y2 was isolated and identified by the internal transcribed spacer of rDNA as Penicillium oxalicum y2. When lecithin, Ca3(PO4)2, or ground phosphate rock were separately used as sole phosphorus source, different phosphate-releasing modes were observed. The strain y2 was able to release as high as 2090 mg/L soluble phosphate within 12 days of incubation with Ca3(PO4)2 as sole phosphorus source. In the culture solution, high concentration of oxalic, citric, and malic acids and high phosphatase activity were detected. The organic acids contributed to solubilizing inorganic phosphate sources, while phosphatase was in charge of the mineralization of organic phosphorus lecithin. Afterwards, the fungus culture was applied to the soil with rape growing. During 50 days of incubation, the soil's available phosphate concentration increased by three times compared with the control, the dry weight of rape increased by 78.73%, and the root length increased by 38.79%. The results illustrated that P. oxalicum y2 possessed both abilities of solubilizing inorganic phosphorus and mineralizing organic phosphorus, which have great potential application in providing biofertilizer for modern agriculture.

Black aspergilli in Brazilian onions: From field to market.

The occurrence of black aspergilli in onions has been reported as frequent, and this group of fungi harbors potentially toxigenic species. In addition, Aspergillus niger has been reported as the causative agent of black mold rot, an important postharvest disease that causes damage throughout the world. Brazil stands out as one of the world's largest onion producers. However, few studies have been conducted to investigate the mycobiota in Brazilian onions. For this reason, we investigated the mycobiota of 48 market (n = 25) and field (n = 23) onion bulb samples. Nineteen soil samples were collected from the same fields and evaluated. In field onions and soil samples, Penicillium spp. was the prevalent fungal group, whereas in market samples A. section Nigri was the most frequent group. Due to the taxonomic complexity of this group, species identification was supported by phylogenetic data (CaM gene). A. welwitschiae was the most prevalent species in market samples. Black aspergillus strains were evaluated for fumonisin B2 (FB2) and ochratoxin A (OTA) production. Overall, 53% and 2.2% of the strains produced FB2 and OTA, respectively. The occurrence of FB2 and OTA was also investigated in onion bulb samples but none showed contamination with these mycotoxins.

Soil fungal biodiversity and pathogen identification of rotten disease in Aconitum carmichaelii (Fuzi) roots.

Aconitum carmichaelii, commonly known as Fuzi, is a typical traditional Chinese medicine (TCM) herb that has been grown for more than one thousand years in China. Although root rot disease has been seriously threatening this crop in recent years, few studies have investigated root rot disease in Fuzi, and no pathogens have been identified. In this study, fungal libraries from rhizosphere soils were constructed by internal transcribed spacer (ITS) sequencing using the HiSeq 2500 high-throughput platform. A total of 948,843 tags were obtained from 17 soil samples, and these corresponded to 195,583,495 nt. At 97% identity, the libraries yielded 12,266 operational taxonomic units (OTUs), of which 97.5% could be annotated. In sick soils, Athelia, Mucor and Mortierella were the dominant fungi, comprising 10.3%, 10.1% and 7.7% of the fungal community, respectively. These fungi showed 2.6-, 1.53- to 6.31- and 1.38- to 2.65-fold higher enrichment in sick soils compared with healthy soils, and their high densities reduced the fungal richness in the areas surrounding the rotted Fuzi roots. An abundance analysis suggested that A. rolfsii and Mucor racemosus, as the dominant pathogens, might play important roles in the invading Fuzi tissue, and Phoma adonidicola could be another pathogenic fungus of root rot. In contrast, Mortierella chlamydospora, Penicillium simplicissimum, Epicoccum nigrum, Cyberlindnera saturnus and Rhodotorula ingeniosa might antagonize root rot pathogens in sick soils. In addition, A. rolfsii was further verified as a main pathogen of Fuzi root rot disease through hypha purification, morphological observation, molecular identification and an infection test. These results provide theoretical guidance for the prevention and treatment of Fuzi root rot disease.

New Penicillium and Talaromyces species from honey, pollen and nests of stingless bees.

Penicillium and Talaromyces species have a worldwide distribution and are isolated from various materials and hosts, including insects and their substrates. The aim of this study was to characterize the Penicillium and Talaromyces species obtained during a survey of honey, pollen and the inside of nests of Melipona scutellaris. A total of 100 isolates were obtained during the survey and 82% of those strains belonged to Penicillium and 18% to Talaromyces. Identification of these isolates was performed based on phenotypic characters and ß-tubulin and ITS sequencing. Twenty-one species were identified in Penicillium and six in Talaromyces, including seven new species. These new species were studied in detail using a polyphasic approach combining phenotypic, molecular and extrolite data. The four new Penicillium species belong to sections Sclerotiora (Penicillium fernandesiae sp. nov., Penicillium mellis sp. nov., Penicillium meliponae sp. nov.) and Gracilenta (Penicillium apimei sp. nov.) and the three new Talaromyces species to sections Helici (Talaromyces pigmentosus sp. nov.), Talaromyces (Talaromyces mycothecae sp. nov.) and Trachyspermi (Talaromyces brasiliensis sp. nov.). The invalidly described species Penicillium echinulonalgiovense sp. nov. was also isolated during the survey and this species is validated here.

A Novel Penicillium sp. Causes Rot in Stored Sugar Beet Roots in Idaho.

Penicillium vulpinum along with a number of other fungi can lead to rot of stored sugar beet roots. However, Penicillium isolates associated with necrotic lesions on roots from a recent sugar beet storage study were determined to be different from P. vulpinum and other recognized Penicillium species. Phylogenies based on sequencing of the internal transcribed spacer (ITS)-5.8S, ß-tubulin (BenA), and RNA polymerase II second largest subunit (RPB2) DNA regions indicate that these isolates are novel, but most closely related to the following Penicillium spp. in the section Fasiculata: P. aurantiogriseum, P. camemberti, and P. freii. Macro- and micromorphological data also support designating these isolates as a new species for which we propose the name, Penicillium cellarum sp. nov. Inoculation studies with the P. cellarum isolates on roots of the commercial sugar beet cultivar B-7 led to the formation of necrotic lesions 23 to 25 mm in diameter after 86 days in storage. These lesions were similar to those observed on sugar beet roots in commercial storage piles. These data indicate that P. cellarum is a pathogen which can cause root rot in stored sugar beet roots.

Taxonomic analysis of the microbial community in stored sugar beets using high-throughput sequencing of different marker genes.

Post-harvest colonization of sugar beets accompanied by rot development is a serious problem due to sugar losses and negative impact on processing quality. Studies on the microbial community associated with rot development and factors shaping their structure are missing. Therefore, high-throughput sequencing was applied to describe the influence of environment, plant genotype and storage temperature (8°C and 20°C) on three different communities in stored sugar beets, namely fungi (internal transcribed spacers 1 and 2), Fusarium spp. (elongation factor-1α gene fragment) and oomycetes (internal transcribed spacers 1). The composition of the fungal community changed during storage mostly influenced by the storage temperature followed by a weak environmental effect. Botrytis cinerea was the prevalent species at 8°C whereas members of the fungal genera Fusarium and Penicillium became dominant at 20°C. This shift was independent of the plant genotype. Species richness within the genus Fusarium also increased during storage at both temperatures whereas the oomycetes community did not change. Moreover, oomycetes species were absent after storage at 20°C. The results of the present study clearly show that rot development during sugar beet storage is associated with pathogens well known as causal agents of post-harvest diseases in many other crops.

Culture and molecular identification of fungal contaminants in edible bird nests.

Widespread food poisoning due to microbial contamination has been a major concern for the food industry, consumers and governing authorities. This study is designed to determine the levels of fungal contamination in edible bird nests (EBNs) using culture and molecular techniques. Raw EBNs were collected from five house farms, and commercial EBNs were purchased from five Chinese traditional medicine shops (companies A-E) in Peninsular Malaysia. The fungal contents in the raw and commercial EBNs, and boiled and unboiled EBNs were determined. Culturable fungi were isolated and identified. In this study, the use of these methods revealed that all EBNs had fungal colony-forming units (CFUs) that exceeded the limit set by Standards and Industrial Research Institute of Malaysia (SIRIM) for yeast and moulds in EBNs. There was a significant difference (p < 0.05) in the number of types of fungi isolated from raw and commercial EBNs, but no significant difference in the reduction of the number of types of fungi after boiling the EBNs (p > 0.05). The types of fungi isolated from the unboiled raw EBNs were mainly soil, plant and environmental fungi, while the types of fungi isolated from the boiled raw EBNs, unboiled and boiled commercial EBNs were mainly environmental fungi. Aspergillus sp., Candida sp., Cladosporium sp., Neurospora sp. and Penicillum sp. were the most common fungi isolated from the unboiled and boiled raw and commercial EBNs. Some of these fungi are mycotoxin producers and cause opportunistic infections in humans. Further studies to determine the mycotoxin levels and methods to prevent or remove these contaminations from EBNs for safe consumption are necessary. The establishment and implementation of stringent regulations for the standards of EBNs should be regularly updated and monitored to improve the quality of the EBNs and consumer safety.

Isolation and identification of toxigenic and non-toxigenic fungi in samples of medicinal plants from the market / Isolamento e identificação de fungos toxigênicos e não toxigênicos em amostras plantas medicinais do comércio

ABSTRACT: The consumption of preparations of medicinal plants has been increasing during the last decades in occidental societies. The presence of toxigenic fungi in a plant product may represent a potential risk of contamination, because of aflatoxins and ochratoxins. In this study, 12 samples of medicinal plants were analyzed in relation to the level of fungal contamination, and the presence of producers of ochratoxin A and aflatoxins was assessed by visualization of fungi using a cromatovisor in coconut milk. Most of the species found belong to the genus Cladosporium, Fusarium, Aspergillus and Penicillium. Species producing ochratoxin A were present in 2 samples (16.7%), Melissa and Hibiscus. Species producing aflatoxin were found in samples of Jacaranda decurrens (8.33%). This study suggests that herbs, if stored improperly, can provide the growth of fungi and should be examined before consumption.

RESUMO: O consumo das plantas medicinais vem aumentando nas últimas décadas nas sociedades ocidentais, porém, a presença de fungos toxigênicos nestas plantas pode representar um risco em potencial de contaminação devido à produção de aflatoxinas e ocratoxinas. Neste trabalho, 12 amostras de plantas medicinais foram analisadas em relação ao nível de contaminação por fungos, enquanto a presença de produtores de ocratoxina A e aflatoxinas foi avaliada pela visualização em cromatovisor dos fungos em meio de leite de coco. A maioria das espécies encontradas pertence aos gêneros Cladosporium, Fusarium, Aspergillus e Penicillium. Espécies produtoras de ocratoxina A estavam presentes em 2 amostras (16,7%), Melissa e Hibisco. Espécies produtoras de aflatoxina foram encontradas na amostra de Carobinha (8,33%). Este trabalho sugere que as ervas, sendo armazenadas inadequadamente, proporcionam o crescimento de fungos e, por isso, estes devem ser examinados antes do consumo.

Transformation of inorganic P fractions of soil and plant growth promotion by phosphate-solubilizing ability of Penicillium oxalicum I1.

The solubilization of tricalcium phosphate is often considered as the standard for screening of most phosphate-solubilizing microorganisms (PSMs). However, usually the effect of large-scale application of PSM on the promotion of crop growth varies. This study presents an efficient method for screening and testing phosphate-solubilizing fungus that enhance plant growth. A fungus Penicillium oxalicum I1 (P-I1) was isolated and identified that had high ability of phosphate-solubilization and could utilize maize root exudates as sources, and propagate well in vitro and in soil. P-I1 excreted oxalic acid and reached 593.9 µg/ml, and the pH value was decreased from 6.90 to 1.65 in 26 h. The amount of P-I1 increased by 48-fold in 28 d and was maintained for 49 d in soil. PSM showed selectivity on the transformation of the different forms of phosphorus, a wide range of insoluble phosphates, such as Ca8H2(PO4)6·5H2O, AlPO4, FePO4, and Ca10(PO4)6(OH)2, were converted to soluble CaHPO4in soil, and CaHPO4was also inhibited from being converted into insoluble phosphate by P-I1. The Ca2-P content reached 27.11 µg/g soil on day 28 at 20°C, which increased by 110.32%, and plant growth promotion was tested and verified, the results showed that maize yield increased remarkably than control after inoculated P-I1, maize yield increased maximum by 14.47%. The data presented that P-I1 appear attractive for exploring their plant growth-promoting activity and potential field application.

Gymnemagenin-producing endophytic fungus isolated from a medicinal plant Gymnema sylvestre R.Br.

Gymnema sylvestre is a plant containing the triterpenoid gymnemagenin, which is used in the pharmaceutical industry as an antidiabetic agent. The objective of this study was to determine whether endophytic fungi, isolated from G. sylvestre, produce gymnemagenin. We isolated an endophytic fungal strain from the leaves of G. sylvestre which produces gymnemagenin in the medium. The fungus was identified as Penicillium oxalicum based on morphological and molecular methods. The strain had a component with the same TLC Rf value and HPLC retention time as authentic gymnemagenin. The presence of gymnemagenin was further confirmed by FTIR, UV, and (1)H NMR analyses.

Fermentation of ginseng extracts by Penicillium simplicissimum GS33 and anti-ovarian cancer activity of fermented products.

A total of 58 isolates of ß-glucosidase-producing microorganisms were isolated from soil around the wild ginseng roots under forest using Esculin-R2A agar. Among these isolates, strain GS33 showed a strong ability to convert ginsenosides Rb1, Rb2, Rc, and Rd into F2, Rg3, C-K, and convert ginsenoside Rg1 into Rh1, and F1. Fermented ginseng products can inhibit ES-2 cells growth and the IC50 value was 0.73 mg ml⁻¹. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain GS33 belongs to the genus Penicillium and is most closely related to Penicillium simplicissimum (99 %).

Microbial biotransformation of gentiopicroside by the endophytic fungus Penicillium crustosum 2T01Y01.

Endophytic fungi are symbiotic with plants and possess multienzyme systems showing promising metabolite potency with region selectivity and stereoselectivity. The aim of this study was to use these special microorganisms as an in vitro model to mimic the potential mammalian metabolites of a natural iridoid gentiopicroside (GPS, compound 1). The fungi isolated from a medicinal plant, Dendrobium candidum Wall. ex Lindl., were screened for their biotransformation abilities with GPS as the substrate, and one strain with high converting potency was identified as Penicillium crustosum 2T01Y01 on the basis of the sequence of the internal transcribed spacer of the ribosomal DNA region. Upon the optimized incubation of P. crustosum 2T01Y01 with the substrate, seven deglycosylated metabolites were detected by ultraperformance liquid chromatography/quadrupole time of flight mass spectrometry (UPLC/Q-TOF MS). Preparative-scale biotransformation with whole cells of the endophytic fungus resulted in the production of five metabolites, including three novel ones, 5α-(hydroxymethyl)-6ß-methyl-3,4,5,6-tetrahydropyrano[3,4-c]pyran-1(8H)-one (compound 2), (Z)-4-(1-hydroxybut-3-en-2-yl)-5,6-dihydropyran-2-one (compound 3), and (E)-4-(1-hydroxybut-3-en-2-yl)-5,6-dihydropyran-2-one (compound 4), along with two known ones, 5α-(hydroxymethyl)-6ß-methyl-1H,3H-5,6-dihydropyrano[3,4-c]pyran-1(3H)-one (compound 5) and 5α-(hydroxymethyl)-6α-methyl-5,6-dihydropyrano[3,4-c]pyran-1(3H)-one (compound 6), aided by nuclear magnetic resonance and high-resolution mass spectral analyses. The other two metabolites were tentatively identified by online UPLC/Q-TOF MS as 5-hydroxymethyl-5,6-dihydroisochromen-1-one (compound 7) and 5-hydroxymethyl-3,4,5,6-tetrahydroisochromen-1-one (compound 8), and compound 8 is a new metabolite. To test the metabolic mechanism, the ß-glucosidase activity of the fungus P. crustosum 2T01Y01 was assayed with ρ-nitrophenyl-ß-d-glucopyranoside as a probe substrate, and the pathway of GPS biotransformation by strain 2T01Y01 is proposed. In addition, the hepatoprotective activities of GPS and metabolite compounds 2, 5, and 6 against human hepatocyte line HL-7702 injury induced by hydrogen peroxide were evaluated.

Isolation and screening of strains producing high amounts of rutin degrading enzymes from Fagopyrum tataricum seeds.

The rutin degrading enzyme (RDE) was isolated and purified from tartary buckwheat seeds. The RDE was purified about 11.34-fold and its final yield was 3.5%, which was very low, due to our purification strategy of giving priority to purity over yield. The RDE molecular weight was estimated to be about 60 kDa. When rutin was used as substrate, an optimal enzyme activity was seen at around pH 5.0 and 40 °C. Strains isolation strategy characterized by the use of rutin as sole carbon source in enrichment cultures was used to isolate RDE-producing strains. Then the active strains were identified by morphology characterization and 18s rDNA-ITS (Internal Transcribed Spacer) gene sequencing. Three isolates coded as B3, W2, Y2 were successfully isolated from fusty Fagopyrum tataricum flour cultures. Strain B3 possessed the highest unit activity among these three strains, and its total activity reached up to 171.0 Unit. The active isolate (B3) could be assigned to Penicillium farinosum. When the Penicillium farinosum strains were added to tartary buckwheat flour cultures at pH 5.0, 30 °C after 5 days fermentation, the quercetin production raised up to 1.78 mg/l, almost 5.1 times higher than the fermentation without the above active strains. Hence, a new approach was available to utilize microorganism-aided fermentation for effective quercetin extraction from Fagopyrum tataricum seeds.

Two new Penicillium species Penicillium buchwaldii and Penicillium spathulatum, producing the anticancer compound asperphenamate.

Penicillium buchwaldii sp. nov. (type strain CBS 117181(T)  = IBT 6005(T)  = IMI 30428(T) ) and Penicillium spathulatum sp. nov. (CBS 117192(T)  = IBT 22220(T) ) are described as new species based on a polyphasic taxonomic approach. Isolates of P. buchwaldii typically have terverticillate conidiophores with echinulate thick-walled conidia and produce the extrolites asperphenamate, citreoisocoumarin, communesin A and B, asperentin and 5'-hydroxy-asperentin. Penicillium spathulatum is unique in having restricted colonies on Czapek yeast agar (CYA) with an olive grey reverse, good growth on CYA supplemented with 5% NaCl, terverticillate bi- and ter-ramulate conidiophores and consistently produces the extrolites benzomalvin A and D and asperphenamate. The two new species belong to Penicillium section Brevicompacta and are phylogenetically closely related to Penicillium tularense. With exception of Penicillium fennelliae, asperphenamate is also produced by all other species in section Brevicompacta (P. tularense, Penicillium brevicompactum, Penicillium bialowiezense, Penicillium olsonii, Penicillium astrolabium and Penicillium neocrassum). Both new species have a worldwide distribution. The new species were mainly isolated from indoor environments and food and feedstuffs. The fact that asperphenamate has been found in many widely different plants may indicate that endophytic fungi rather than the plants are the actual producers.

Bioproduction of bile acids and the glycine conjugates by Penicillium fungus.

It has been found that the Penicillium endophytic filamentous fungus with the young stems of Scurrula atroprupurea (Loranthaceae) produces cholic acid, deoxycholic acid and the glycine conjugates.

Degradation of mikan (Japanese mandarin orange) peel by a novel Penicillium species with cellulolytic and pectinolytic activity.

AIMS: The mikan, or Japanese mandarin orange, is a popular fruit in Japan, but its peel is one of the major agricultural wastes. The aims of this study were to screen, isolate, and characterize a mikan peel-degrading microbe. METHODS AND RESULTS: Several samples including activated sludge, sediment, compost and spoiled mikan peel were collected and cultured in a minimal salt medium containing mikan peel as the sole carbon source. Degradation activity was found in a culture of the spoiled mikan peel, and a fungal strain, designated OP1, with both cellulolytic and pectinolytic activity was isolated. No toxic metabolites, such as mycotoxins, were found in OP1 cultures, as evaluated by gas chromatography/mass spectrometry. A phylogenetic analysis strongly suggested that OP1 is a novel species of the genus Penicillium. CONCLUSIONS: Results suggest that Penicillium sp. OP1 plays an important role in aerobic microbial degradation of cellulose/pectin-rich biomasses in soil ecology, and further imply that this strain may be useful for both simultaneous cellulase/pectinase production and reduction of agricultural waste. SIGNIFICANCE AND IMPACT OF THE STUDY: The present results advance our understanding of microbial degradation of cellulose/pectin-rich biomasses in the natural environment, and offer a new tool for reduction of agricultural waste, which is important for sustaining circulatory societies.

Development and application of an assay for uranyl complexation by fungal metabolites, including siderophores.

An assay to detect UO(2)(2+) complexation was developed based on the chrome azurol S (CAS) assay for siderophores (B. Schwyn and J. B. Neilands, Anal. Biochem. 160:47-56, 1987) and was used to investigate the ability of fungal metabolites to complex actinides. In this assay the discoloration of two dyed agars (one containing a CAS-Fe(3+) dye and the other containing a CAS-UO(2)(2+) dye) caused by ligands was quantified. The assay was tested by using the siderophore desferrioxamine B (DFO), and the results showed that there was a regular, reproducible relationship between discoloration and the amount of siderophore added. The ratio of the discoloration on the CAS-UO(2)(2+) agar to the discoloration on the CAS-Fe(3+) agar was independent of the amount of siderophore added. A total of 113 fungi and yeasts were isolated from three soil samples taken from the Peak District National Park. The fungi were screened for the production of UO(2)(2+) chelators by using the CAS-based assay and were also tested specifically for hydroxamate siderophore production by using the hydroxamate siderophore auxotroph Aureobacterium flavescens JG-9. This organism is highly sensitive to the presence of hydroxamate siderophores. However, the CAS-based assay was found to be less sensitive than the A. flavescens JG-9 assay. No significant difference between the results for each site for the two tests was found. Three isolates were selected for further study and were identified as two Pencillium species and a Mucor species. Our results show that the new assay can be effectively used to screen fungi for the production of UO(2)(2+) chelating ligands. We suggest that hydroxamate siderophores can be produced by mucoraceous fungi.