Guttation droplets of <Emphasis Type="Italic">Penicillium nordicum</Emphasis> and <Emphasis Type="Italic">Penicillium verrucosum</Emphasis> contain high concentrations of the mycotoxins ochratoxin A and B

Eight of eleven ochratoxigenic isolates of Penicillium nordicum and Penicillium verrucosum produced guttation droplets when grown on Czapek yeast extract (CYA) agar for 10–14 days at 25°C. Parallel cultivation of one strain each of P. nordicum and P. verrucosum on malt extract agar demonstrated that higher volumes of exudate are produced on this agar. However, HPLC analyses revealed higher concentrations of ochratoxin A (OTA) and B (OTB) in droplets originating from cultures on CYA. For quantitative determination of the mycotoxin contents, triplicates of three isolates each of P. nordicum and P. verrucosum were grown as single spot cultures on CYA for up to 14 days at 25°C. Guttation droplets were carefully collected between day 11 and 14 with a microliter syringe from each culture. Extracts from exudates and corresponding mycelia as well as fungal free agar were analyzed by HPLC for the occurrence of ochratoxin A (OTA) and B (OTB). Mean concentrations ranging between 92.7–8667.0 ng OTA and 159.7–2943.3 ng OTB per ml were detected in the guttation fluids. Considerably lower toxin levels were found in corresponding samples of the underlying mycelia (9.0–819.3 ng OTA and 4.5–409.7 ng OTB/g) and fungal free agar (15.3–417.0 ng OTA and 12.7–151.3 ng OTB/g). This is the first report which shows that high amounts of mycotoxins could be excreted from toxigenic Penicillium isolates into guttation droplets.     

Growth and mycotoxin production by <Emphasis Type="Italic">Chaetomium globosum</Emphasis>

Chaetomium globosum, the most common species within this genus, produces chaetoglobosins A and C when cultured on building material. Relatively low levels of these compounds have been shown to be lethal to various tissue culture cell lines. This study had two major objectives: (1) to determine the frequency at which Chaetomium species are isolated in water-damaged buildings and (2) to examine the production of chaetoglobosins A and C in isolates of C. globosum obtained from different buildings. Out of 794 water-damaged buildings, Chaetomium species were isolated in 49% of these structures. C. globosum ATCC 16021 was grown on four different media: oatmeal agar (OA), potato dextrose agar (PDA), corn meal agar (CMA), and malt extract agar (MEA). After 4 weeks, fungal growth was evaluated based on colony diameter and the quantity of spores produced on agar plates. In addition, production of chaetoglobosin A and C was monitored using high performance liquid chromatography. Colony diameter, spore production, and mycotoxin production by C. globosum were the highest on OA. Out of 30 C. globosum isolates cultured on OA for 4 weeks, 16 produced detectable amounts of chaetoglobosin A and every isolate produced chaetoglobosin C.     

Production of moniliformin by Canadian isolates of Fusarium

Twenty-eight Canadian isolates of Fusarium were tested for their ability to produce moniliformin in corn. Both F. moniliforme (2/6 isolates) and F. subglutinans (11/15 isolates) produced the mycotoxin, while F. graminearum did not. Field-corn inoculated with F. moniliforme M3783 was able to support production of both moniliformin and fusarin C.     

Impact of aggressiveness of <Emphasis Type="Italic">Fusarium graminearum</Emphasis> and <Emphasis Type="Italic">F. culmorum</Emphasis> isolates on yield parameters and mycotoxin production in wheat

Plant-associated isolates from Fusarium graminearum and F. culmorum were inoculated on wheat in field experiments in 2007 and 2008 to ascertain their influence on fungal colonization of the ears, as well as mycotoxin contamination (deoxynivalenol, DON; nivalenol, NIV; zearalenone, ZEA) and yield parameters in the mature crop after inoculation with or without irrigation. The isolates were assigned to four different groups of aggressiveness on the basis of pathogenic symptom development and mycotoxin production in vitro. Increased levels of trichothecene-producing Fusarium DNA in the ears indicated a successful inoculation of the plants, which resulted in increased DON content in the wheat kernels in 2007. Dry conditions at anthesis markedly suppressed fungal colonization as well as mycotoxin accumulation. However, due to precipitation during the ripening period, yield and thousand-kernel weight were similar whether or not irrigation was applied at the time of inoculation. The level of aggressiveness among the isolates as determined in vitro was not reflected in the field experiment. The activity of the extracellular invertase in developing ears increased as a plant response to pathogen infection, especially when the plants were irrigated at the time of inoculation. In 2008, the Fusarium inoculation of wheat heads did not cause fungal growth and mycotoxin contamination in the grain, because of the dry weather conditions that occurred over the entire period of anthesis and ripening. The risk of future mycotoxin contamination in grains was discussed based on climate change prognosis.     

Aflatoxin and Ochratoxin Production by <Emphasis Type="Italic">Aspergillus</Emphasis> Species Under Ex Vivo Conditions

Aspergillus species are increasingly important human pathogens. It is not known whether toxic metabolites of many of these pathogenic species can act as virulence factors in aspergillosis. We examined isolates of aflatoxin and ochratoxin-producing species for toxin production in ex vivo conditions. Seven of the 21 aflatoxin-producing isolates screened produced aflatoxin at 35 and 37°C on the general medium yeast extract sucrose agar (YES). However, none of them produced toxin at these temperatures on brain heart infusion agar (BHA), a medium that mimics human tissue, or on BHA with modified pH or sugar levels. Six of the 12 ochratoxin-producing isolates examined produced toxin at 35°C on YES. All three isolates of A. alliaceus produced ochratoxin on BHA or modified BHA at 37°C. One strain of A. pseudoelegans produced a minute amount of ochratoxin on modified BHA at 37°C. These data indicate that aflatoxin is an unlikely virulence, factor but that ochratoxin may be a potential virulence factor in aspergillosis.     

Production of patulin and citrinin by <Emphasis Type="Italic">Penicillium expansum</Emphasis> from British Columbia (Canada) apples

Twenty-four isolates of Penicillium expansum Link from British Columbia (Canada) apples were cultured in yeast-extract sucrose (YES) at 25°C for 28 days to investigate production of patulin and citrinin. These isolates proved to be potent producers of citrinin, patulin, or in most cases, both mycotoxins. In every isolate, citrinin, patulin, or both compounds were produced at levels as high as 565 μg/mL (mean 269 μg/mL) and 100 μg/mL (mean 31 μg/mL), respectively. Of the 24 isolates, 4 produced citrinin only, and 2 produced patulin only. Overall, 83% of the isolates formed patulin and 91% formed citrinin. YES broth proved to be an effective medium for patulin and citrinin production. Other workers have noted that production of these mycotoxins in culture often presages production in fruits, so these results might help Canadian fruit processors evaluate and minimize mycotoxin levels in their products.     

Assessment of aflatoxin and cyclopiazonic acid production by Aspergillus flavus isolates from Hungary

Thirty-two isolates of Aspergillus flavus were obtained from various sources in Hungary. All isolates were morphologically identified as A. flavus and three atypical variants were confirmed as A. flavus by comparing their DNA with an ex type culture of A. flavus. None of these isolates produced aflatoxins when tested on coconut agar or grown on rice medium and culture extracts examined by thin layer chromatography. Also, none of the isolates converted sterigmatocystin, O-methyl sterigmatocystin, norsolorinic acid, or sodium acetate to aflatoxin. However, 59% of the isolates produced cyclopiazonic acid based on thin layer chromatographic analysis of culture extracts. The isolates that lack the ability to produce both aflatoxin and cyclopiazonic acid are potential candidates for use in bicontrol studies.     

Enniatin B and ochratoxin A in the blood serum of workers from the waste management setting

The waste management occupational environment is recognized by the simultaneous presence of several substances and biologic agents. Therefore, workers are exposed simultaneously to multiple contaminants. Occupational exposure to aflatoxin B₁ in one Portuguese waste sorting plant was already reported. However, besides this mycotoxin, data regarding fungal contamination showed that exposure to other mycotoxins could be expected. A study was developed to analyze if exposure to other mycotoxins besides aflatoxin B₁ was occurring in the workers from the waste sorting plant previously assessed and to discuss how these findings need to be considered in the risk assessment process. In addition to aflatoxin B₁ detected previously by ELISA, two additional mycotoxins and one mycotoxin degradation product were detected and quantified by a multi-mycotoxin HPLC-MS/MS approach: Enniatin B and ochratoxin A as well as 2’R-ochratoxin A. Besides the confirmation of co-exposure to several mycotoxins, results probably indicate different exposure routes for the mycotoxins reported.     

Early discrimination of fungal species responsible of ochratoxin A contamination of wine and other grape products using an electronic nose

An electronic nose (e-nose) system using an array of metal oxide sensors (Fox 3000, Alpha MOS) was used to detect and discriminate two ochratoxigenic fungal species, Aspergillus carbonarius (Bain.) Thom and A. niger Van Tieghem, that are responsible for the contamination of wine and other wine grape products, using their volatile production patterns. Two well-known ochratoxigenic strains were used in this study: A. carbonarius A941 and A. niger A75. These strains were grown on three culture media, Czapek Dox modified (CDm) agar, yeast extract sucrose (YES) agar and white grape juice (WGJ) agar, and the volatile organic compounds produced in the headspace by these species were evaluated over periods of 48–120 h. The e-nose system was able to differentiate between the two species within 48 h of growth on YES and WGJ agar using principal component analysis (PCA), which accounted for 99.9% and 97.2% of the data respectively, in principal components 1 and 2, based on the qualitative volatile profiles. This differentiation was confirmed by cluster analysis of data. However, it was not possible to separate these species on CDm agar. Our results show that the two closely related ochratoxigenic species responsible for the contamination of wine and other wine grape products can be discriminated by the use of qualitative volatile fingerprints. This approach could have potential for rapid identification of A. carbonarius and A. niger on wine grape samples, thereby significantly reducing the time of detection of these ochratoxin A producing species.     

Influence of carbon source on growth and mycotoxin production by isolates of Pyrenophora tritici-repentis from wheat

The fungus Pyrenophora tritici-repentis can infect wheat kernels, causing red smudge, and has been shown to produce the anthraquinone mycotoxins emodin, catenarin, and islandicin. The growth of 8 fungal isolates from diverse regions was evaluated on various culture media and was found to be generally slowest on the semisynthetic Fries medium. The choice of carbon source had a significant effect on mycotoxin production, as assessed by high-performance liquid chromatography. The highest emodin concentration (194.18 ± 16.26 μg/g medium) was observed for isolate Alg 3-24 on Fries medium supplemented with fructose, while the highest catenarin concentration (302.54 ±13.92 μg/g medium) was observed for TS93-71B on Fries medium supplemented with starch. Islandicin was not produced by any isolate under the conditions tested. Evaluation of the dynamics of mycotoxin production by isolate 331-2 on V8-potato dextrose agar medium revealed a rapid accumulation of emodin and catenarin during the first week of incubation, followed by a large decline by 14 days. Differences in the growth of and mycotoxin production by isolates of P. tritici-repentis likely resulted from the differential composition of the media and (or) intraspecies variability. Accordingly, the optimization of growth medium should be considered when evaluating the potential of specific isolates for mycotoxin production.     

Fusarium crookwellense,a newly isolated fungus from wheat in Japan: Its mycotoxin production and pathogenicity to wheat and barley

Fusarium crookwellense was isolated for the first time in Japan from scabby wheat harvested in Hokkaido in 1991. Four isolates were obtained and examined for their mycological features on culture media, mycotoxin production, and pathogenicity to wheat and barley. The texture of fungal colonies and the morphology of macroconidia on a potato dextrose agar medium were similar to those ofFusarium graminearum. AllF. crookwellense isolates examined produced nivalenol, 4-acetylnivalenol, and zearalenone on rice media at levels ranging from 0.9 to 22.5 µg/g, 0.5 to 25.0 µg/g, and 1.4 to 162.5 µg/g, respectively. All were pathogenic toward the wheat and barley strains tested, and scab symptoms were found on the heads and leaves of plants. This is the first report on domestic isolates ofF. crookwellense from the crop field in Japan.     

Biodegradation of aliphatic hydrocarbon by indigenous fungi isolated from used motor oil contaminated sites

Eighteen indigenous fungal isolates has been successfully isolated from samples of used motor oil, top five centimetres of soil and drainage water contaminated with used motor oil. All of the pure fungal isolates obtained were identified, characterized and subjected to preliminary screening by evaluating the average growth rate of each fungal isolates on minimal media containing 1% (v/v) used motor oil. Trichoderma asperellum strain TUB F-1067 (SA4), Trichoderma asperellum strain Tr48 (SA5), Trichoderma asperellum strain TUB F-756 (SA6), Penicillium species (P1), and Aspergillus species (P9) were further selected for their hydrocarbon biodegradation potential. Among these five fungal isolates selected, P1 strain presented a significant degree of degradation by degrading almost all of the n-alkanes (n-C-₁₅ to n-C-₂₃ range) present in the used motor oil, thus of greater potential in degrading the aliphatic hydrocarbon compounds of used motor oil. The authors would like to certify that the work have not been sent/considered to be published in other journals.     

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.     

Oxidase testing from Kligler's iron agar and triple sugar iron agar slants

Many members of the familyVibrionaceae have been implicated as causative agents of diarrhea. Most of these organisms are non-lactose fermenters, and all are oxidase-positive. If the oxidase test could be reliably performed on growth from the surface of Kligler's iron agar and/or triple sugar iron agar slants, it would aid in the screening of potential stool pathogens. Forty-six isolates from the generaAeromonas, Plesiomonas, andVibrio were inoculated onto Kligler's iron agar and triple sugar iron agar slants, incubated overnight, and tested for oxidase activity. All 46 isolates produced alkaline over acid, with or without gas, Kligler's iron agar slants and were oxidase-positive. On triple sugar iron agar slants, 13 isolates produced these same patterns, and all were oxidase-positive. Acid over acid, with gas, triple sugar iron agar slants were produced by 18 isolates, and all were oxidase-positive. Acid over acid, without gas, triple sugar iron agar slants were produced by 15 isolates, and all were oxidase-negative. These negative oxidase tests were due to low pH. Oxidase tests performed from the surface of Kligler's iron agar and triple sugar iron agar slants used to screen stool isolates were reliable, provided the slants were acid over acid with gas, or alkaline over acid with or without gas. Kligler's iron agar is recommended with this procedure, since most potential stool pathogens of both theEnterobacteriaceae and theVibrionaceae will produce an alkaline over acid, with or without gas, slant, and false negative oxidase tests will be minimized.     

Novel and rapid agar plate methods for in vitro assessment of bacterial biocontrol isolates’ antagonism against multiple fungal phytopathogens

Biological control of plant diseases with antagonistic bacteria is a promising alternative to conventional chemical control strategies. In vitro screening for inhibition of mycelial growth of phytopathogenic fungi by bacterial isolates is the first step in selecting putative bacterial biocontrol agents. Dual culture plate assay is the most common method involved in this first-line selection process. However, it needs independent agar plates to test antagonism by a specific bacterial isolate against each of the fungal phytopathogen. Two modified in vitro antagonism tests are proposed here. Antagonistic activity of a putative biocontrol bacterial strain against four different fungal phytopathogens could be assessed in a single agar plate simultaneously. A comparison of the new methods with conventional dual culture plate assay was also done. The proposed methods are easy to perform and results of antagonism are obtained rapidly. Results of fungal inhibition were qualitatively comparable with that generated through dual culture plate assay. Quantity of resources such as agar medium and plates required for the modified antagonistic assays is several folds less than that required for dual culture plate assay.     

Aflatoxin B1 producing potential of isolates of Aspergillus flavus Link ex Fries from cotton,maize and wheat

Twenty-one isolates ofAspergillus flavus Link ex Fries obtained from cotton, maize and wheat were screened for their ability to produce aflatoxins on two liquid media. Of these, sixteen isolates were toxigenic and produced only aflatoxin B₁ as assessed by bioassay on okra seedlings and TLC method. For screening isolates ofA. flavus for aflatoxin formation, 0.7 % YES+ Salt medium was found to be good as also for obtaining higher yields of the toxin. Isolates ofA. flavus produced aflatoxin B₁ ranging from 0.85 to 17.2 mg/50 ml. Maximum yield of aflatoxin was obtained when rice was used as the substrate in case of toxigenic isolates L-27 and C-9, and on maize in isolate M-11.     

Cyclopiazonic acid production by aflatoxigenic and non-aflatoxigenic strains of aspergillus flavus

Twenty-eight of 54 isolates of Aspergillus flavus grown on autoclaved agricultural commodities such as wheat, rice and corn were found to produce the mycotoxin cyclopiazonic acid. Eighteen of the A. flavus isolates produced aflatoxin, and fourteen isolates produced both cyclopiazonic acid and aflatoxin. A preliminary screening of some aflatoxin-contaminated corn samples revealed for the first time the natural occurrence of cyclopiazonic acid in agricultural commodities.     

Visual clone identification of Penicillium commune isolates

A method for visual clone identification of Penicillium commune isolates was developed. The method is based on images of fungal colonies acquired after growth on a standard medium and involves a high degree of objectivity, which in future studies will make it possible for non-experts to perform a qualified identification of different species as well as clones within a species. A total of 77 P. commune isolates from a cheese dairy were 3-point inoculated on Yeast Extract Sucrose (YES) agar and incubated for 7 days at 25 degrees C. After incubation, the isolates were classified into groups containing the same genotype determined by DNA fingerprinting (AFLP). Each genotype also has a specific phenotype such as different colony colours. By careful image acquisition, colours were measured in a reproducible way. Prior to image analysis, each image was corrected with respect to colour, geometry and self-illumination, thereby gaining a set of directly comparable images. A method for automatic extraction of a given number of concentric regions was used. Using the positions of the regions, a number of relevant features--capturing colour and colour-texture from the surface of the fungal colonies--was extracted for further analysis. We introduced the Jeffreys-Matusitas (JM) distance between the feature distributions to express the similarity between regions in two colonies, and to evaluate the overall (weighted) similarity. The nearest neighbour (NN) classification rule was used. On a dataset from 137 isolates, we obtained a "leave-one-out" cross-validation identification rate of approximately 93-98% compared with the result of DNA fingerprinting.     

A comparative study of Phytophthora meadii isolates from rubber (Hevea brasiliensis) plantations in Sri Lanka

Thirty-three Phytophthora meadii isolates were obtained from different Hevea brasiliensisclones grown in different climatic regions in Sri Lanka. Growth, morphology and pathogenicity of the isolates were compared. Growth and pathogenicity levels varied among the isolates. Some isolates obtained from moderately susceptible rubber clones were highly pathogenic, compared to isolates obtained from resistant clones. Highly pathogenic isolates produced a higher number of sporangia on agar at 27 ± 2°C. It was impossible to group isolates according to the clone or the region from where they were obtained.This revised version was published online in October 2005 with corrections to the Cover Date.