Dual fungal infection with Lomentospora prolificans and Aspergillus fumigatus: A pathogenetic diagnosis elucidated after two decades by sequential examinations.

A 44-year-old male Japanese was admitted for further post-remission treatments for acute myeloid leukemia. He developed a right orbital abscess. An isolate of Lomentospora prolificans was obtained from the lesion, and orbital biopsy also revealed the presence of Aspergillus fumigatus. This fatal case involved a concurrent dual fungal infection.

Microflora of Mycotoxigenic Fungi in Rice Grains in Kyushu Region of Japan and Their Changes during Storage under non-Controlled Conditions.

Contamination of agricultural crops by mycotoxins has increased because of the expansion of mycotoxin-producing fungi along with global warming. In this study, the fungal microflora of brown rice grains cultivated in Kyushu region in the southern part of Japan was investigated. A total of 75% of rice samples examined in this study showed less than 30% of fungal contamination rates with a median rate of 12.5%. Some isolates of Aspergillus flavus showed the ability to produce aflatoxins (AFs) (AFB1 production was 62.5-70.4 ng/mL) . Furthermore, AF-producing Aspergillus flavus survived during storage and Aspergillus creber, which produced sterigmatocystin, was detected in a stored rice sample. Although AFs or sterigmatocystin-contamination was not detected in any rice samples, these mycotoxin-producing fungi are distributed and can survive during storage under the natural conditions in Japan. Employing suitable storage conditions is important for preventing mycotoxin contamination of brown rice grains.

Occurrence of Penicillium brocae and Penicillium citreonigrum, which Produce a Mutagenic Metabolite and a Mycotoxin Citreoviridin, Respectively, in Selected Commercially Available Rice Grains in Thailand.

Commercially available rice grains in Thailand were examined to isolate the monoverticillate Penicillium species responsible for toxic yellowed rice. Penicillium species were obtained from seven out of 10 rice samples tested. Among them, one Penicillium citreonigrum isolate and six Penicillium brocae isolates were morphologically identified. The P. citreonigrum isolate produced the mycotoxin citreoviridin on a yeast extract sucrose broth medium. Mycotoxin surveys showed that citreoviridin was not detected in any samples, but one out of 10 rice samples tested was positive for aflatoxin B1 at a level of 5.9 µg/kg. An Ames test revealed that methanol extracts from rice grains inoculated with selected P. brocae isolates were positive for strains TA100 and YG7108 of Salmonella typhimurium, suggesting the presence of base-pair substitution and DNA alkylation mutagens. Our data obtained here demonstrated that aflatoxin B1 and toxic P. citreonigrum were present on domestic rice grains in Thailand, although limited samples were tested. Penicillium brocae, which may produce mutagenic metabolites, was isolated for the first time from the surface of Thai rice grains.

Experimental infection of Fusarium proliferatum in Oryza sativa plants; fumonisin B1 production and survival rate in grains.

Fusarium proliferatum is a plant pathogenic fungus associated with crops such as asparagus and corn, and it possesses the ability to produce a range of mycotoxins, including fumonisins. In Asia, rice (Oryza sativa) is a staple cereal and is occasionally colonized by this fungus without obvious physiological changes. F. proliferatum is closely related to Gibberella fujikuroi (anamorph F. fujikuroi) responsible for Bakanae disease in rice; however there are few reports of F. proliferatum as a rice pathogen. In this study, we examined the pathogenic potential of F. proliferatum in rice plants with respect to browning, fumonisin production, and survival rates in rice grains. Fungal inoculation was conducted by spraying a conidial suspension of F. proliferatum onto rice plants during the flowering period. Browning was found on the stalk, leaf, and ear of rice. Fumonisin B(1) was detected at levels from trace to 21 ng/g grains, using tandem mass spectrometry. Fungal recovery after 6 months indicated that F. proliferatum had high affinity to rice plants being still viable in grains. From this study, it can be concluded that F. proliferatum is a possible pathogen of rice and possesses a potential to produce fumonisin B(1) in rice grains in the field.

[Application of DNA extraction kit, 'GM quicker' for detection of genetically modified soybeans].

Several DNA extraction methods have been officially introduced to detect genetically modified soybeans, but the choice of DNA extraction kits depend on the nature of the samples, such as grains or processed foods. To overcome this disadvantage, we examined whether the GM quicker kit is available for both grains and processed foods. We compared GM quicker with four approved DNA extraction kits in respect of DNA purity, copy numbers of lectin gene, and working time. We found that the DNA quality of GM quicker was superior to that of the other kits for grains, and the procedure was faster. However, in the case of processed foods, GM quicker was not superior to the other kits. We therefore investigated an unapproved GM quicker 3 kit, which is available for DNA extraction from processed foods, such as tofu and boiled soybeans. The GM quicker 3 kit provided good DNA quality from both grains and processed foods, so we made a minor modification of the GM quicker-based protocol that was suitable for processed foods, using GM quicker and its reagents. The modified method enhanced the performance of GM quicker with processed foods. We believe that GM quicker with the modified protocol is an excellent tool to obtain high-quality DNA from grains and processed foods for detection of genetically modified soybeans.

Fusarium verticillioides strains isolated from corn feed: characterization by fumonisin production and RAPD fingerprinting

In this study a total of 16 Fusarium verticillioides strains isolated from corn feed samples were characterized by fumonisin (FB) production and random amplified polymorphic DNA (RAPD). All the strains produced FB1 and FB2 with levels ranging from 2.41 to 3996.36 µg/g, and from 1.18 to 1209.91 µg/g, respectively. From the 16 F. verticillioides strains, four were identified as low (3.59 to 1289.84 µg/g), eight as intermediate (>1289.84 to 3772.44 µg/g) and four strains as high (>3772.44 µg/g) fumonisin producers. From the total of 105 loci amplified, 60 (57.14 percent) were polymorphic. RAPD analysis showed very similar patterns among low, moderate and high fumonisin-producing strains. Although RAPD markers were capable of discriminating the different F. verticillioides strains, there was no clear association between these makers and fumonisin production.

Neste estudo, 16 cepas de F. verticillioides isoladas de amostras de ração de milho foram caracterizadas com base na produção de fumonisinas (FB) e em marcadores de polimorfismos de DNA amplificado ao acaso (RAPD). Todas as cepas produziram FB1 e FB2, com níveis variando, respectivamente, de 2,41 a 3996,36 µg/g e 1,18 a 1209,91 µg/g. De acordo com a produção de fumonisinas totais (FB1 + FB2) e a distribuição por análise de quartis, do total de 16 cepas de F. verticillioides, quatro foram identificadas como baixas produtoras de fumonisinas (3,59 a 1289,84 µg/g), oito como intermediárias (>1289,84 a 3772,44 µg/g) e quatro como altas produtoras de fumonisinas (>3772,44 µg/g). Os 10 primers utilizados amplificaram 105 locos, 60 (57,14 por cento) dos quais foram polimórficos. As análises de RAPD mostraram padrões muito similares entre as cepas baixas, médias e altas produtoras de fumonisinas. Embora os marcadores RAPD tenham se mostrado capazes de discriminar as diferentes cepas de F. verticillioides, não foi detectada nenhuma associação entre estes marcadores e a produção de fumonisinas.

Arthrographis kalrae, a rare causal agent of onychomycosis, and its occurrence in natural and commercially available soils.

We report a rare case of onychomycosis due to Arthrographis kalrae, and describe an attempt to elucidate the source of the infection by fungal isolation and PCR-based methods for detection of A. kalrae. The patient was a 63-year-old Japanese man, whose fingernails, excluding the right index finger, were markedly dystrophic. Microscopic examination of his fingernails disclosed hyaline hyphae and arthroconidia. The causal agent was identified as A. kalrae based on morphological characteristics and ITS region sequencing. The fungus was susceptible in vitro to terbinafine, itraconazole, and miconazole. A therapeutic trial of oral terbinafine, 125 mg/day and topical 1% miconazole for seven months brought about a complete cure of the ungual lesion. We found that A. kalrae could be detected at 1.1 x 10(4) CFU/g (92.4% incidence) in soil from the patient's garden. Furthermore, varying CFUs of A. kalrae were recovered in 61% of the samples obtained from crop field soils, commercially available soils or allied products for gardening. Since the patient was working with soil that harbored A. kalrae, the possibility of acquiring the infection from this source should not be excluded.

Mycotoxicological quality evaluation of corn samples used by processing industries in the Northern region of Paraná State, Brazil.

Based on fungal and fumonisin contamination of 870 freshly harvested samples, the quality of corn used by processing industries in the Northern region of Parana State, Brazil (2003 and 2004 crop-year) was evaluated. Sampling was carried out for each crop at two points in the production chain, i.e. at reception by the processors and at the pre-drying step. Corn samples were more frequently contaminated with Fusarium sp. (100%) and Penicillium sp. (84.1-95.3%) than Aspergillus sp. (5.6-19.8%). Fumonisin B(1) (FB(1)) was detected in all samples from the two points in both crop-years. FB(1) levels ranged 0.02-11.83 microg g(-1) in the reception and 0.02-10.98 microg g(-1) in the pre-drying samples of the 2003 crop. Samples from the 2004 crop showed FB(1) levels ranging 0.03-12.04 microg g(-1) in the reception and 0.06-7.74 microg g(-1) in the pre-drying samples. FB(2) levels ranged 0.02-5.25 microg g(-1) in the reception and 0.01-7.89 microg g(-1) in the pre-drying samples (2003 crop-year). In samples from the 2004 crop, FB(2) levels ranged 0.02-6.12 microg g(-1) in the reception and 0.05-3.47 microg g(-1) in the pre-drying samples. Low fumonisin levels were detected in most corn samples used by processors in the Northern region of Parana State, showing a decreasing trend in fumonisin contamination over the years.

Immunotoxicity of nivalenol after subchronic dietary exposure to rats.

Immunobiological effects of nivalenol (NIV), a trichothecene mycotoxin produced by Fusarium nivale, were examined in male F344 rats after 90-day dietary exposure at doses of 0, 0.4, 1.5, and 6.9 mg/kg body weight/day (0, 6.25, 25 and 100 ppm, respectively) in a subchronic toxicity study. With regards to the serum immunoglobulin levels, a slight increase of IgM was observed only at 6.9 mg/kg (26% increase), while levels of IgG and IgA did not fluctuate at any dose. Flow cytometric analysis of splenic cells revealed a dose-dependent decrease of T lymphocyte/B lymphocyte (CD3(+)/B220(+)) ratio from 1.5mg/kg and an elevated CD4(+)helper/CD8(+)cytotoxic T lymphocyte ratio at 6.9 mg/kg. Furthermore, increases of natural killer (NK) activity of splenic lymphocytes against YAC-1 target cells were observed at all doses, while the magnitude of changes was similar between 1.5 and 6.9 mg/kg. At 6.9 mg/kg, the reduction of the ratio of NKR-P1A(+) splenic cells, which is an indicator of NK cells in the spleen, was apparent. As with other previous studies of NIV, decreased body weight was observed from 1.5 mg/kg during the experiment in the present study. In summary, NIV affected immune function in rats after 90-day dietary exposure, the effects being apparent from 0.4 mg/kg judging from the increase of NK activity, although nutritional suppression might have influenced the immunological changes appeared from 1.5mg/kg.

Fumonisins in corn: correlation with Fusarium sp. count, damaged kernels, protein and lipid content

A contaminação natural por fungos e fumonisinas foi avaliada em 109 amostras de milho recém-colhido do Estado do Paraná e correlacionada com grãos ardidos (%). Além disso, grãos sadios e ardidos de 24 amostras de milho foram selecionados a fim de comparar o perfil da microbiota fúngica e níveis de fumonisinas. A correlação entre os teores de proteínas/lipídios e os níveis de fumonisinas também foi analisada nos 15 híbridos de milho mais freqüentemente cultivados no Estado do Paraná. A contagem total de fungos em 109 amostras de milho recém-colhido variou de 1,9x104 a 3,5x106 UFC/g, Fusarium sp. de 1,0x103 a 2,2x106 UFC/g e, níveis de fumonisinas de 0,13 a 20,38 µg/g. A contagem total de fungos/Fusarium spp. e níveis de fumonisinas apresentaram correlação positiva (p<0,05). Adicionalmente, houve uma correlação positiva entre grãos ardidos (%) e a contagem total de fungos/ Fusarium spp. (p < 0,05). Os níveis de fumonisinas nos grãos sadios variaram de 0,57 a 20,38 µg/g, enquanto que nos grãos ardidos variaram de 68,96 a 336,38 µg/g. Não foi observada correlação significativa entre os níveis de fumonisinas e os teores de proteínas/lipídios. Esses resultados ratificam a importância do monitoramento constante da contaminação por fungos toxigênicos e fumonisinas em milho e derivados a fim de garantir a qualidade e segurança dos produtos e minimizar o risco potencial à saúde humana e animal.

[Evaluation of three commercial ELISA kits for rapid screening of deoxynivalenol in unpolished wheat].

Evaluation of commercial ELISA kits for the screening of deoxynivalenol (DON) was carried out. Three kinds of commercial kits supplied by different companies were used. Three lots of naturally contaminated wheat and DON-free wheat (<0.05 microg/g) were used as samples. The values obtained from ELISA were compared with those of the HPLC-UV method. In the results obtained from 14 institutions using ELISA, the CV values were less than 17.6% for all the commercial kits. The DON-free sample was not detected as positive with any commercial kits. Also there was no negative finding among the naturally contaminated samples used in this experiment. Coefficients of determination in ELISA and HPLC analysis were 0.979-0.999. These results indicate that ELISA using any of the three commercial kits is efficient for the screening of DON in wheat.

Trends of fumonisin contamination and animal intoxication through monitoring 1991 to 1997 corn crop in the State of Paraná, Brazil.

Eleven feed samples associated with six animal (horse and poultry) intoxication outbreaks (1991) in the state of Paraná, Brazil, were evaluated for fungal and fumonisin contamination. In order to estimate the trend of livestock intoxication, fumonisin contamination was monitored in corn produced both at the commercial level (1991, 1995 crop), and in an experimental field at a local Agronomy Institute (1997 crop). The total mould count in the feed samples ranged from 2.9 x 10(3) to 1.9 x 10(7) CFU/g, with Fusarium verticillioides as the predominant species, at a high count of 2.4 x 10(4)-6.5 x 10(5) CFU/g. Fumonisins (FB1 + FB2) were detected in all corn-based feed samples at levels ranging from 2.89 to 14.54 microg/g. All 27 Northern corn samples (1991 crop) were contaminated with fumonisins at levels ranging from 2.32 to 16.64 microg/g. Twenty-six (96.3%) out of 27 corn samples from the Central-Southern region (1995 crop) were positive for fumonisins (FB1+FB2), with the range of 0.07-3.66 microg/g, while all 37 Northern samples (1995 crop) were contaminated with fumonisins ranging from 0.57 to 9.97 microg/g. Twenty-one out of 37 corn samples from the Northern region (1997 crop) were positive for fumonisins, but at low level (range of 0.05-2.67 microg/g). The results showed a decreasing trend in fumonisin contamination over the years. Nowadays animal intoxication outbreaks rarely occur in this State, as both animal producers and feed industries have become conscious about monitoring of corn and other raw materials at the quality control level.

Remarkable influence of the aromatic substructure in 9-methoxystrobilurin derivatives on their antifungal activity.

9-Methoxystrobilurin-type beta-methoxyacrylate antibiotics (MOSBs) having various aromatic substructures were synthesized. The antifungal activity of the synthesized MOSBs against pathogenic and non-pathogenic fungi was examined, and the obtained results revealed that the antifungal activity of MOSBs was highly dependent on the aromatic substructures. However, no significant correlation was observed between cytotoxicity against human fibroblasts-like cell line and their structural properties. In addition, our results suggested that the strong growth-inhibitory activity of 9-methoxystrobilurin K against human-derived cell lines should be related to its hindered ether-type substructure.