Mycoviruses diversity in the black kōji mold, Aspergillus luchuensis (section Nigri) isolated from liquor-production environments in Japan.

Koji mold, which belongs to the Aspergillus section Nigri, is used in the production of shochu. The section Nigri is composed of very morphologically similar members that in some cases produce mycotoxins, which rises concerns as to whether the presence of mycotoxin-producing fungi in shochu producing sites can compromise consumer safety. Thus, we examined the presence of mycotoxin-producing sec. Nigri fungi in six shochu factories (named A–F) in Japan. Airborne fungal levels in the factories were determined, and a traditional koji called “kona-koji” made from the mold naturally present in factory C (Aogashima village) was analyzed. Isolates of sec. Nigri fungi were identified morphologically and confirmed via cytochrome b gene analysis. In factory A (Nago city), airborne fungal levels of sec. Nigri were 4,000 and 100 cfu/m3 in the koji-making and fermentation rooms, respectively. In factories B, C, and D, the levels were 40, >104 cfu/m3, and 100 cfu/m3, respectively. In factory F (Iki city), there were high levels of airborne white-koji mold (a white mutant of Asp. luchuensis). The most dominant fungal species of sec. Nigri was isolated and identified as Asp. luchuensis via genetic analysis. This is likely to have originated from the commercial fermentation culture used. Asp. niger and Asp. luchuensis were isolated from kona-koji. Mycotoxin production (ochratoxin and fumonisin B2) by Asp. luchuensis (eight strains) and Asp. niger (three strains) was virtually inexistent; only one strain of Asp. niger was positive for fumonisin B2. This study clearly shows that mycotoxin-producing fungi are not dominant in the fungal flora present in the shochu factories examined and therefore, that the liquor can be safely fermented. Implications: In this study, we examined the presence of mycotoxin-producing Aspergillus sec. Nigri fungi in six shochu (Japanese distilled beverage) factories. The most dominant fungal species of sec. Nigri was isolated and identified as Aspergillus luchuensis (black-koji mold). The proportion of mycotoxin-producing Aspergillus niger and Aspergillus carbonarius was very small. In addition, the Asp. niger isolated from koji mold did not have the ability to produce ochratoxins or fumonisin B. This study clearly shows that shochu can be safely fermented.

Ochratoxin A and related fungi in Brazilian black pepper (Piper nigrum L.)

Soil is a reservoir for Aspergillus flavus and A. parasiticus, fungi that commonly colonize peanut seeds and produce carcinogenic aflatoxins. Densities of these fungi in soil vary greatly among fields and may influence the severity of peanut infection. This study examined the relationship between soil density of Aspergillus species and the incidence of peanut seed colonization under laboratory conditions. Viable peanut seeds were wounded and inoculated with 20 soils differing in composition and density of Aspergillus species and were then incubated for 14 days at 37 degrees C (seed water activity = 0.92). The effect of soil density of individual section Flavi species (A. flavus strains L and S, A. parasiticus, A. caelatus, and A. tamarii), section Nigri, and A. terreus on the incidence of seed colonization was best expressed as a function of exponential rise to maximum. Exponential curves often rose to maximum percentages of seed colonization by section Flavi species that were well below 100% despite high species densities in some soils. Competition primarily among section Flavi species may explain the reduced incidences of seed colonization. An average of two or fewer propagules of each Aspergillus species in the soil at the wound site was required for colonization of 20% of peanut seeds. Other fungal species were capable of invading peanut seeds only when soil densities of sections Flavi and Nigri species were low.

The study aimed to determine the fungal diversity in clinical waste samples from a healthcare facility in Penang Malaysia. Different fungi species were detected in 83.75% of the 92 clinical waste samples that were screened from different sections of the healthcare facility. One hundred fifty fungal isolates comprising of 8 genera and 36 species were obtained. They were purified by using single spore isolation technique. Subsequently, the isolates were identified by phenotypic method based on morphological and culture characteristics on different culture media. Among all fungal isolates, Aspergillus spp. in section Nigri 10.2%, Aspergillus niger 9.5%, Aspergillus fumigatus 8.8%, Penicillium. simplicissium 8%, Aspergillus tubingensis 7.3%, Aspergillus terreus var. terreus 6.6%, Penicillium waksmanii 5.9% and Curvularia lunata 6.5% were the most frequent. Among five sections of the Wellness Centre, the clinical wastes collected from the diagnostic labs of haematology section had the highest numbers of fungal species (29 species). Glove wastes had the highest numbers of fungal species (19 species) among 17 types of clinical wastes screened. Among all fungal species, Aspergillus spp. exhibited higher growth at 37°C than at 28°C, indicating the potential of these opportunistic fungi to cause diseases in human. These results indicated the potential of hospital wastes as reservoirs for fungal species.

<p><strong><em>testing of different isolation method of microorganisms from ginger rhizomes.</em></strong></p><p>Common method of isolation of pathogenic fungi from the rhizome of ginger is direct isolation planting. However, a modification is some time needed to find pathogenic and saphrophytic fungi in the rhizome. Nine midified methods of isolation were tested in the isolation of fungi from the rhizome of ginger collected from market and field, by using PDA medium. It was showed that by wasting diseased rhizomes, more species of fungi were isolated and the isolates grew more rapidly Particullarly storage fungi from rhizomes collected from market. Species of fungi were found in this study such as <em>Cephalosporium </em>sp., <em>Trichoderma</em> sp., <em>Penicillium</em> sp., <em>Mucor</em> sp., <em>Aspergillus flavus</em>, <em>Aspergillus</em> sp., <em>Chalaropsis </em>sp., <em>Gliocladium </em>sp<em>., Rhizopus</em> sp., <em>Gloeosporium</em> sp., <em>Fusarium oxysporum</em>, <em>F. moniliformae, F. equiseti, Fusarium </em>sp.</p>

In 2012 to 2014, Philippine green coffee beans from Coffea arabica in Benguet and Ifugao; Coffea canephora var. Robusta in Abra, Cavite, and Ifugao; and Coffea liberica and Coffea excelsea from Cavite were collected and assessed for the distribution of fungi with the potential to produce ochratoxin A (OTA). The presence of fungal species was evaluated both before and after surface sterilization. There were remarkable ecological and varietal differences in the population of OTA-producing species from the five provinces. Aspergillus ochraceus, A. westerdijkiae, and Penicillium verruculosum were detected from Arabica in Benguet and Ifugao while Aspergillus carbonarius, Aspergillus niger, and Aspergillus japonicus were isolated in Excelsa, Liberica, and Robusta varieties from Abra, Cavite, and Davao. Contamination by Aspergillus and Penicillium species was found on 59 and 19 %, respectively, of the 57 samples from five provinces. After disinfection with 1% sodium hypochlorite, the levels of infection by Aspergillus and Penicillium fell to 40 and 17%, respectively. A total of 1184 fungal isolates were identified to species level comprising Aspergillus sections Circumdati (four species), Clavati (one), Flavi (one), Fumigati (one), Nigri (three), and Terrie (one). Within section Circumdati, 70% of A. ochraceus produced OTA as high as 16238 ng g(-1) while 40% of A. westerdijkiae produced maximum OTA of 36561 ng g(-1) in solid agar. Within section Nigri, 16.76% of A. niger produced OTA at the highest 18439 ng g(-1), 10% of A. japonicus at maximum level of 174 ng g(-1), and 21.21% of A. carbonarius yielded maximum OTA of 1900 ng g(-1). Of the 12 species of Penicillium isolated, P. verruculosum was ochratoxigenic, with a maximum OTA production of 12 ng g(-1).

Fungi in gorgonians are now known to cause gorgonian diseases, but little attention has been paid to the nature of fungal communities associated with gorgonians. The diversity of culturable fungi associated with six species of healthy South China Sea gorgonians were investigated using a culture-dependent method followed by analysis of fungal internal transcribed spacer sequences. A total of 121 fungal isolates were recovered and identified using the Basic Local Alignment Search Tool search program. These belonged to 41 fungal species from 20 genera. Of these, 30 species and 12 genera are new reports for gorgonians, and the genera Aspergillus and Penicillium were the most diverse and common in the six gorgonian species. Comparison of the fungal communities in the six gorgonian species, together with results from previous relevant studies, indicated that different gorgonian species and the same gorgonian species living in different geographic locations had different fungal communities. The gorgonian Dichotella gemmacea harbored the most fungal species and isolates, while Echinogorgia aurantiaca had the least fungal diversity. Among the six media used for fungal isolation, potato glucose agar yielded the highest isolates (27 isolates), while glucose peptone starch agar had the best recoverability of fungal species (15 species). The antimicrobial activity of the 121 fungal isolates was tested against three marine bacteria and two marine gorgonian pathogenic fungi. A relatively high proportion (38%) of fungal isolates displayed distinct antibacterial and antifungal activity, suggesting that the gorgonian-associated fungi may aid their hosts in protection against pathogens. This is the first report comparing the diversity of fungal communities among the South China Sea gorgonians. It contributes to our knowledge of gorgonian-associated fungi and further increases the pool of fungi available for natural bioactive product screening.

The traditional Japanese single distilled liquor, which uses koji and yeast with designated ingredients, is called “honkaku shochu.” It is made using local agricultural products and has several types, including barley shochu, sweet potato shochu, rice shochu, and buckwheat shochu. In the case of honkaku shochu, black koji fungus (Aspergillus luchuensis) or white koji fungus (Aspergillus luchuensis mut. kawachii) is used to (1) saccharify the starch contained in the ingredients, (2) produce citric acid to prevent microbial spoilage, and (3) give the liquor its unique flavor. In order to make delicious shochu, when cultivating koji fungus during the shochu production process, we use a unique temperature control method to ensure that these three important elements, which greatly affect the taste of the produced liquor, are balanced without any excess or deficiency. This review describes in detail the production method of honkaku shochu, a distilled spirit unique to Japan and whose market is expected to expand worldwide, with special attention paid to the koji fungi cultivation step. Furthermore, we describe the history of the koji fungi used today in the production of shochu, and we provide a thorough explanation of the characteristics of each koji fungi. We also report the latest research progress on this topic.

We use a set of three resistive sensors based on undoped multi-walled carbon nanotubes, B-doped multi-walled carbon nanotubes, and N-doped multi-walled carbon nanotubes to study fungal infection in strawberries inoculated with Rhizopus sp. or with Aspergillus sp. section Nigri. We apply tristimulus analysis using the conductance variation of the sensors when exposed to the infected strawberries to distinguish between uninfected strawberries and strawberries infected with Rhizopus sp. or with Aspergillus sp. section Nigri, and to obtain a graphical representation providing a tool for the simple and fast detection and identification of the fungal infection.

Diversity of Aspergillus section Nigri on the surface of Vitis labrusca and its hybrid grapes

To investigate the prevalence of fungal agents in local Egyptian broiler breeder chicken’s premises, tracheal and cloacal swabs from chickens, feed, and water samples were collected. The targeted breed's dead-in-shell eggs and newly hatched chick’s samples were also tested. All fungal isolates were morphologically typed and the predominant fungal species were further subjected to molecular typing using PCR-RFLP and gene sequence analysis of the β-tubulin gene. Results revealed a high prevalence of fungal isolates in tracheal and cloacal swabs (39.3 - 48.1%) and feed and water samples (37.5% and 28.6%, respectively). Fungal isolation rates in dead-in-shell eggs of all breeds were high except in Dahaby breed. Aspergillus species including A. niger, A. flavus, and A. terreus were the predominantly isolated fungi from all collected samples. The s-Tubulin genes PCR-RFLP of selected Aspergillus isolates showed a characteristic restriction pattern for each species; however, the method was unable to distinguish between strains. The β-tubulin gene phylogenetic and sequence analysis of selected A. flavus, and A. terreus from breeder chickens and their hatching chicks indicated their relatedness to isolates from bronchopulmonary Aspergillosis in humans in the Middle East. In conclusion, the Aspergillus species remains the most prevalent fungi in breeder chickens, their incubated eggs and hatched chicks indicating their widespread in hatcheries. The PCR-RFLP is an easy tool to discriminate between Aspergilli species, however, the β-tubulin sequence analysis more descriptive of potential sources of fungal contamination. Further epidemiological studies are needed to monitor avian and human Aspergilli in poultry houses with a special focus on antifungal drug-resistant strains.

Effect of pepA deletion and overexpression in Aspergillus luchuensis on sweet potato shochu brewing

Otomycosis is a fungal infection of the external auditory canal caused mainly by the genus Aspergillus. Aspergillus luchuensis, an industrially important fungus, is a member of Aspergillus section Nigri. In this report, we present a case of otomycosis due to Aspergillus luchuensis in a 43-year-old female patient. We performed a partial PCR-sequencing of β-tubulin and calmodulin genes to identify the isolate to the species level. Further, we determined the in vitro susceptibility of the isolate to nystatin, clotrimazole and itraconazole according to the Clinical & Laboratory Standards Institute (CLSI) M38-A2 protocol. Accordingly, the minimum inhibitory concentrations of clotrimazole, nystatin and itraconazole were 0.25μg/mL, 0.5μg/mL and 1μg/mL, respectively. This is the first report of clinically relevant isolation of Aspergillus luchuensis identified by a molecular technique as a causative agent of otomycosis.

The peanut plant accumulates defensive stilbenoid phytoalexins in response to the presence of soil fungi, which in turn produce phytoalexin-detoxifying enzymes for successfully invading the plant host. Aspergillus spp. are opportunistic pathogens that invade peanut seeds; most common fungal species often produce highly carcinogenic aflatoxins. The purpose of the present research was to evaluate the in vitro dynamics of peanut phytoalexin transformation/detoxification by important fungal species. This work revealed that in feeding experiments, Aspergillus spp. from section Flavi were capable of degrading the major peanut phytoalexin, arachidin-3, into its hydroxylated homolog, arachidin-1, and a benzenoid, SB-1. However, Aspergillus niger from section Nigri as well as other fungal and bacterial species tested, which are not known to be involved in the infection of the peanut plant, were incapable of changing the structure of arachidin-3. The results of feeding experiments with arachidin-1 and resveratrol are also reported. The research provided new knowledge on the dynamics of peanut stilbenoid transformations by essential fungi. These findings may contribute to the elucidation of the phytoalexin detoxification mechanism involved in the infection of peanut by important toxigenic Aspergillus spp.

The fruits of organically and conventionally produced coffee are subject to contamination from several species of fungi, and that may be related to poor beverage quality and mycotoxin production. The aim of this study was to identify the biodiversity of isolated filamentous fungi in the coffee beans produced on organic and conventional farms within the same area. Two hundred and twelve isolates belonging to eleven different genders were identified from the fifteen samples analyzed. The main gender found was Aspergillus, with isolation of fungi from the Sections Circumdati, Nigri, Flavi and Versicolores. The samples that obtained the greatest index of contamination were those that didn't pass through the disinfection process with 1% sodium hypochlorite. The samples of coffee beans from organic cultivation exhibited the greatest degree of richness and diversity within a single location with very similar climatic conditions. Thus, organic coffee production requires greater care due to the increased incidence of filamentous fungi.