Deep Sequencing, Nested PCR, and Denaturing Gradient Gel Electrophoresis Reveal a Wider Distribution of Chinese Caterpillar Mushroom, Ophiocordyceps sinensis (Ascomycetes), in Native Soil Types.

Ophiocordyceps sinensis appears as stroma emerging from underground sclerotium enclosed by the skeleton of Thitarodes moth larvae. However, the actual distribution of the fungus in soil still remains unclarified. In this study, 40 soil samples were used for detection of O. sinensis to confirm its distribution in native habitats using denaturing gradient gel electrophoresis, nested internal transcribed spacer (ITS) PCR, and 454 pyrosequencing methods. The soil samples included six types: Os, where both stromata and host moth larvae were found; NL, representing no signs of stromata, but where moth larvae were found; NOs, where neither stroma nor moth larvae were found; BS, with bare soil without the presence of stroma of O. sinensis or moth larvae; AF, from soil surrounding the stroma; and MP, soil particles firmly wrapping the sclerotium of O. sinensis. Of 40 samples tested, 36 showed positive detection of O. sinensis by at least one of the three detection methods, with positive detection in all six sample types at all five sites. The results showed that traces of O. sinensis can be detected in locations with no macroscopically visible evidence of the fungus or its host and at least 100 m away from such locations.

Combined bacterial and fungal targeted amplicon sequencing of respiratory samples: Does the DNA extraction method matter?

BACKGROUND: High-throughput sequencing techniques are used to analyse the diversity of the respiratory microbiota in health and disease. Although extensive data are available regarding bacterial respiratory microbiota, its fungal component remains poorly studied. This is partly due to the technical issues associated with fungal metagenomics analyses. In this study, we compared two DNA extraction protocols and two fungal amplification targets for combined bacterial and fungal targeted amplicon sequencing analyses of the respiratory microbiota. METHODS: Six sputa, randomly selected from routine samples in Mondor Hospital (Creteil, France) and treated anonymously, were tested after bacterial and fungal routine culture. Two of which were spiked with Aspergillus Fumigati and Aspergillus Nigri (105 conidia/mL). After mechanical lysis, DNA was extracted using automated QIAsymphony® extraction (AQE) or manual PowerSoil® MoBio extraction (MPE). DNA yield and purity were compared. DNA extracted from spiked sputa was subjected to (i) real-time PCR for Aspergillus DNA detection and (ii) combined metagenomic analyses targeting barcoded primers for fungal ITS1 and ITS2, and bacterial V1-V2 and V3-V4 16S regions. Amplicon libraries were prepared using MiSeq Reagent V3 kit on Illumina platform. Data were analysed using PyroMIC© and SHAMAN software, and compared with culture results. RESULTS: AQE extraction provided a higher yield of DNA (AQE/MPE DNA ratio = 4.5 [1.3-11]) in a shorter time. The yield of Aspergillus DNA detected by qPCR was similar for spiked sputa regardless of extraction protocol. The extraction moderately impacted the diversity or relative abundances of bacterial communities using targeted amplicon sequencing (2/43 taxa impacted). For fungi, the relative abundances of 4/11 major taxa were impacted and AQE results were closer to culture results. The V1-V2 or V3-V4 and ITS1 or ITS2 targets assessed similarly the diversity of bacterial and fungal major taxa, but ITS2 and V3-V4 detected more minor taxa. CONCLUSION: Our results showed the importance of DNA extraction for combined bacterial and fungal targeted metagenomics of respiratory samples. The extraction protocol can affect DNA yield and the relative abundances of few bacterial but more fungal taxa. For fungal analysis, ITS2 allowed the detection of a greater number of minor taxa compared with ITS1.

Dead or alive: sediment DNA archives as tools for tracking aquatic evolution and adaptation.

DNA can be preserved in marine and freshwater sediments both in bulk sediment and in intact, viable resting stages. Here, we assess the potential for combined use of ancient, environmental, DNA and timeseries of resurrected long-term dormant organisms, to reconstruct trophic interactions and evolutionary adaptation to changing environments. These new methods, coupled with independent evidence of biotic and abiotic forcing factors, can provide a holistic view of past ecosystems beyond that offered by standard palaeoecology, help us assess implications of ecological and molecular change for contemporary ecosystem functioning and services, and improve our ability to predict adaptation to environmental stress.

Diversity and antimicrobial activity of endophytic fungi isolated from Securinega suffruticosa in the Yellow River Delta.

Securinega suffruticosa (Pall.) Rehd is an excellent natural secondary shrub in the Shell Islands of Yellow River Delta. The roots of S. suffruticosa have high medicinal value and are used to treat diseases, such as neurasthenia and infant malnutrition. Any organism that is isolated from this species is of immense interest due to its potential novel bioactive compounds. In this research, the distribution and diversity of culturable endophytic fungi in S. suffruticosa were studied, and the endophytic fungi with antimicrobial activity were screened. A total of 420 endophytic fungi isolates were obtained from the S. suffruticosa grown in Shell Islands, from which 20 genera and 35 species were identified through morphological and internal transcribed spacer (ITS) sequence analyses. Chaetomium, Fusarium, Cladosporium, and Ceratobasidium were the dominant genera. The high species richness S (42), Margalef index D' (5.6289), Shannon-Wiener index H' (3.1000), Simpson diversity index Ds (0.9459), PIE index (0.8670), and evenness Pielou index J (0.8719) and a low dominant index λ (0.0541) indicated the high diversity of endophytic fungi in S. suffruticosa, the various species of endophytic fungi with obvious tissue specificity. The inhibition percentages of the 12 species of such endophytic fungi against Colletotrichum siamense were 3.6%-26.3%. C. globosum, Fusarium sp.3, and C. ramotenellum had a high antibacterial activity against Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. The Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) were between 0.5 mg/mL and 2 mg/mL. Alkaloid content detection indicated that endophytic fungi had a high alkaloid content, whereas the alkaloid contents of C. globosum and Fusarium sp.3 reached 0.231% and 0.170%, respectively. Members belonging to the endophytic fungal community in the S. suffruticosa of Shell Islands that may be used as antagonists and antibacterial agents for future biotechnology applications were identified for the first time.

A simple and effective method to discern the true commercial Chinese cordyceps from counterfeits.

The Chinese cordyceps, a complex of the fungus Ophiocordyceps sinensis and its species-specific host insects, is also called "DongChongXiaCao" in Chinese. Habitat degradation in recent decades and excessive harvesting by humans has intensified its scarcity and increased the prices of natural populations. Some counterfeits are traded as natural Chinese cordyceps for profit, causing confusion in the marketplace. To promote the safe use of Chinese cordyceps and related products, a duplex PCR method for specifically identifying raw Chinese cordyceps and its primary products was successfully established. Chinese cordyceps could be precisely identified by detecting an internal transcribed spacer amplicon from O. sinensis and a cytochrome oxidase c subunit 1 amplicon from the host species, at a limit of detection as low as 32 pg. Eleven commercial samples were purchased and successfully tested to further verify that the developed duplex PCR method could be reliably used to identify Chinese cordyceps. It provides a new simple way to discern true commercial Chinese cordyceps from counterfeits in the marketplace. This is an important step toward achieving an authentication method for this Chinese medicine. The methodology and the developmental strategy can be used to authenticate other traditional Chinese medicinal materials.

Characterization and evaluation of mycosterol secreted from endophytic strain of Gymnema sylvestre for inhibition of α-glucosidase activity.

Endophytic fungi produce various types of chemicals for establishment of niche within the host plant. Due to symbiotic association, they secrete pharmaceutically important bioactive compounds and enzyme inhibitors. In this research article, we have explored the potent α-glucosidse inhibitor (AGI) produced from Fusarium equiseti recovered from the leaf of Gymnema sylvestre through bioassay-guided fraction. This study investigated the biodiversity, phylogeny, antioxidant activity and α-glucosidse inhibition of endophytic fungi isolated from Gymnema sylvestre. A total of 32 isolates obtained were grouped into 16 genera, according to their morphology of colony and spores. A high biodiversity of endophytic fungi were observed in G. sylvestre with diversity indices. Endophytic fungal strain Fusarium equiseti was identified through DNA sequencing and the sequence was deposited in GenBank database (https://ncbi.nim.nih.gov) with acession number: MF403109. The characterization of potent compound was done by FTIR, LC-ESI-MS and NMR spectroscopic analysis with IUPAC name 17-(5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a] phenanthren-3-ol. The isolated bioactive compound showed significant α-amylase and α-glucosidase inhibition activity with IC50 values, 4.22 ± 0.0005 µg/mL and 69.72 ± 0.001 µg/mL while IC50 values of acarbose was 5.75 ± 0.007 and 55.29 ± 0.0005 µg/mL respectively. This result is higher in comparison to other previous study. The enzyme kinetics study revealed that bioactive compound was competitive inhibitor for α-amylase and α-glucosidase. In-silico study showed that bioactive compound binds to the binding site of α-amylase, similar to that of acarbose but with higher affinity. The study highlights the importance of endophytic fungi as an alternative source of AGI (α-glucosidase inhibition) to control the diabetic condition in vitro.

Phylogenetic Analysis of the Complete rRNA Gene Sequence of Nosema sp. SE Isolated from the Beet Armyworm Spodoptera exigua.

The microsporidium Nosema sp. SE is a pathogen that infects the beet armyworm Spodoptera exigua. The complete sequence of its 4,302-base pair (bp) ribosomal ribonucleic acid (rRNA) gene region was obtained by polymerase chain reaction amplification and sequencing. The rRNA organization of Nosema sp. SE was 5'-large subunit (LSU) rRNA-internal transcribed spacer-small subunit (SSU) rRNA-intergenic spacer-5S-3', which corresponded to the pattern of Nosema bombycis. Phylogenetic analysis based on LSU rRNA and SSU rRNA both indicated that the parasite had a close relationship with other true Nosema species, confirming that Nosema sp. SE belongs to true Nosema group of the genus Nosema.

Towards a rapid identification and a novel proteomic analysis for dermatophytes from human and animal dermatophytosis.

BACKGROUND: Since accurate identification of dermatophyte species is essential for epidemiological studies and implementing antifungal treatment, overcoming limitations of conventional diagnostics is a fruitful subject. OBJECTIVES AND METHODS: In this study, we investigated real-time polymerase chain reaction(q-PCR), matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS) and nano-electrospray ionisation mass spectrometry (nano-ESI-MS) to detect and identify the most frequently isolated dermatophytes from human and animal dermatophytosis in comparison with conventional methods. RESULTS: Among 200 samples, the identified species were Microsporum canis (78.22%), Trichophyton verrucosum (10.89%) and T. mentagrophytes (5.94%). Q-PCR assay displayed great execution attributes for dermatophytes detection and identification. Using MALDI-TOF MS, M. canis, but none of T. violacium, T. verrucosum or T. mentagrophytes, could be identified. Nano-ESI-MS accurately identified all species. The potential virulence attributes of secreted proteases were anticipated and compared between species. Secreted endoproteases belonging to families/subfamilies of metalloproteases, subtilisins and aspartic protease were detected. The analysed exoproteases are aminopeptidases, dipeptidyl peptidases and carboxypeptidases. Microsporum canis have three immunogenic proteins, siderophore iron transporter mirB, protease inhibitors, plasma membrane proteolipid 3 and annexin. CONCLUSION: In essence, q-PCR, MALDI-TOF MS and nano-ESI-MS assays are very nearly defeating difficulties of dermatophytes detection and identification, thereby, supplement or supplant conventional diagnosis of dermatophytosis.

Occurrence of oleaginous yeast from mangrove forest in Thailand.

A total of 191 yeasts were isolated from 197 samples collected from eight estuarine mangrove forests along four different coastlines of Thailand (Andaman Sea and the East, North and West coasts of the Gulf of Thailand). Of these, 178 isolates were identified as 32 species in 16 genera of Ascomycota, 12 species in nine genera of Basidiomycota, and 13 isolates as potential new species, respectively. Mangroves located along the Andaman Sea coastline had a higher yeast diversity at the species and genera levels than those along the Gulf of Thailand. Kluyveromyces siamensis was the most frequently isolated species, whilst Candida tropicalis was the only species isolated at all eight sites. Screening isolated yeast strains belonging to genera previously reported as oleaginous yeast plus the 13 potential new species, revealed two oleaginous strains, Rhodotorula sphaerocarpa 11-14.4 and Saitozyma podzolica 11-11.3.1. Both of these strains were isolated from the same mangrove forest on the Andaman Sea coastline. They could accumulate lipid when suspended in glucose solution without any supplementation, while the fatty acid composition and oil profile of Rh. sphaerocarpa 11-14.4 and Sait. podzolica 11-11.3.1 were similar to vegetable oil and cocoa butter, respectively.

Combined bacterial and fungal intestinal microbiota analyses: Impact of storage conditions and DNA extraction protocols.

BACKGROUND: The human intestinal microbiota contains a vast community of microorganisms increasingly studied using high-throughput DNA sequencing. Standardized protocols for storage and DNA extraction from fecal samples have been established mostly for bacterial microbiota analysis. Here, we investigated the impact of storage and DNA extraction on bacterial and fungal community structures detected concomitantly. METHODS: Fecal samples from healthy adults were stored at -80°C as such or diluted in RNAlater® and subjected to 2 extraction protocols with mechanical lysis: the Powersoil® MoBio kit or the International Human Microbiota Standard (IHMS) Protocol Q. Libraries of the 12 samples targeting the V3-V4 16S and the ITS1 regions were prepared using Metabiote® (Genoscreen) and sequenced on GS-FLX-454. Sequencing data were analysed using SHAMAN (http://shaman.pasteur.fr/). The bacterial and fungal microbiota were compared in terms of diversity and relative abundance. RESULTS: We obtained 171869 and 199089 quality-controlled reads for 16S and ITS, respectively. All 16S reads were assigned to 41 bacterial genera; only 52% of ITS reads were assigned to 40 fungal genera/section. Rarefaction curves were satisfactory in 3/3 and 2/3 subjects for 16S and ITS, respectively. PCoA showed important inter-individual variability of intestinal microbiota largely overweighing the effect of storage or extraction. Storage in RNAlater® impacted (downward trend) the relative abundances of 7/41 bacterial and 6/40 fungal taxa, while extraction impacted randomly 18/41 bacterial taxa and 1/40 fungal taxon. CONCLUSION: Our results showed that RNAlater® moderately impacts bacterial or fungal community structures, while extraction significantly influences the bacterial composition. For combined bacterial and fungal intestinal microbiota analysis, immediate sample freezing should be preferred when feasible, but storage in RNAlater® remains an option under unfavourable conditions or for concomitant metatranscriptomic analysis; and extraction should rely on protocols validated for bacterial analysis, such as IHMS Protocol Q, and including a powerful mechanical lysis, essential for fungal extraction.

Genetic Variability of the Medicinal Tinder Bracket Polypore, Fomes fomentarius (Agaricomycetes), from the Asian Part of Russia.

We analyzed intraspecies genetic variability of the medicinal tinder bracket polypore, Fomes fomentarius, from the Asian part of Russia, including the Ural, Altai, Western Sayan, and Baikal regions. We used nuclear ribosomal DNA internal transcribed spacer (ITS) sequence data as a standard marker for fungal DNA barcoding. In the Asian part of Russia, lineage A occurs as sublineage A2, which differs from sublineage A1 by a single nucleotide insertion at ITS2.3. Sublineage A2 is distributed up to Lake Baikal in the Ural, Altai, and Western Sayan regions. It can be characterized as a Eurasian sublineage of F. fomentarius. Lineage B is also represented by 2 sublineages (B1 and B2), which differ from each other by nucleotide sequences at ITS2.1. Sublineage B1 is represented by a small group of isolates from Asia (Iran, China, Nepal, South Korea), whereas sublineage B2 mainly includes isolates from Europe (Great Britain, Italy, Latvia, Slovakia, Slovenia) and 2 separate samples from Asia (Iran, China); these locales compose the distribution area of F. fomentarius. In the Asian part of Russia, lineage B is represented by sublineage B2 found in the Southern Urals (at the border between Europe and Asia), which is the only area where sublineages A2 and B2 are present. These sublineages are characterized by different substrate spectra: sublineage A2 is predominantly associated with Betula spp. and rarely with Alnus and Larix trees, whereas sublineage B2 does not have a pronounced substrate preference and is found in basidiomes collected from Acer, Duschekia, Prunus, and Salix trees, but not Betula trees. In general, the spectrum of substrates for F. fomentarius lineages A and B in the Asian part of Russia corresponds to that in other parts of this polypore's distribution area. Data are needed on genetic intraspecies variability (polymorphism) in relation to pharmacological properties for further biotechnological cultivation and use of the medicinal fungus F. fomentarius.

Comparison of various RNA extraction methods, cDNA preparation and isolation of calmodulin gene from a highly melanized isolate of apple leaf blotch fungus Marssonina coronaria.

Marssonina coronaria causes apple blotch disease resulting in severe premature defoliation, and is distributed in many leading apple-growing areas in the world. Effective, reliable and high-quality RNA extraction is an indispensable procedure in any molecular biology study. No method currently exists for RNA extraction from M. coronaria that produces a high quantity of melanin-free RNA. Therefore, we evaluated eight RNA extraction methods including manual and commercial kits, to yield a sufficient quantity of high-quality and melanin-free RNA. Manual methods used here resulted in low quality and black colored RNA pellets showing the presence of melanin, despite all the modifications employed to original procedures. However, these methods when coupled with clean up resulted in melanin-free RNA. On the other hand, all commercial kits used were able to yield high-quality melanin-free RNA having variable yields. TRIzol™ Reagent + RNA Clean & Concentrator™-5 and Ambion-PureLink® RNA Mini Kit were found to be the best methods as the RNA extracted with these methods from 15 day old fungal culture grown on solid medium were free of melanin with good yield. RNA extracted by this improved methodology was applied for RT-PCR, subsequent PCR amplification, and isolation of calmodulin gene sequences from M. coronaria and infected apple leaf pieces. These methods are more time effective than traditional methods and take only an hour to complete. To our knowledge, this is the first report on the method of isolation of high-quality RNA for cDNA synthesis as well as isolation of the calmodulin gene sequence from this fungus.

[Development and evaluation of a rapid PCR detection kit for Ophiocordyceps sinensis].

Ophiocordyceps sinensis is a valuable traditional Chinese medicine. Due to resource shortage, expensive price and huge market demand, there are many adulterants of O. sinensis in markets. Therefore, it is necessary to establish a rapid and effective method for distinguishing O. sinensis. Based on the species-specific PCR of O. sinensis, this study developed a detection kit by optimizing the components and evaluated the specificity, detection limit, repeatability and shelf life of the kit. The results showed that when the quality of O. sinensis accounted for more than 1/200 of that mixture, it could be detected successfully. Moreover, only O. sinensis could be amplified and glowed bright green fluorescence under ultraviolet light. The kit was still in effect when it was placed at 37 ℃ for three days, which indicated that it was stable and effective for one year stored in 4 ℃. The kit in the same batch under different operation conditions, and in different batch under the same operation conditions gave the same result and accuracy, which showed good repeatability of the kit. It is simple, rapid and accurate to distinguish O. sinensis from its adulterants using the kit, and lays the foundation for commercialization of traditional Chinese medicine fast detection kit.

Evaluation of multifarious plant growth promoting traits, antagonistic potential and phylogenetic affiliation of rhizobacteria associated with commercial tea plants grown in Darjeeling, India.

Plant growth promoting rhizobacteria (PGPR) are studied in different agricultural crops but the interaction of PGPR of tea crop is not yet studied well. In the present study, the indigenous tea rhizobacteria were isolated from seven tea estates of Darjeeling located in West Bengal, India. A total of 150 rhizobacterial isolates were screened for antagonistic activity against six different fungal pathogens i.e. Nigrospora sphaerica (KJ767520), Pestalotiopsis theae (ITCC 6599), Curvularia eragostidis (ITCC 6429), Glomerella cingulata (MTCC 2033), Rhizoctonia Solani (MTCC 4633) and Fusarium oxysporum (MTCC 284), out of which 48 isolates were antagonist to at least one fungal pathogen used. These 48 isolates exhibited multifarious antifungal properties like the production of siderophore, chitinase, protease and cellulase and also plant growth promoting (PGP) traits like IAA production, phosphate solubilization, ammonia and ACC deaminase production. Amplified ribosomal DNA restriction analysis (ARDRA) and BOX-PCR analysis based genotyping clustered the isolates into different groups. Finally, four isolates were selected for plant growth promotion study in two tea commercial cultivars TV-1 and Teenali-17 in nursery conditions. The plant growth promotion study showed that the inoculation of consortia of these four PGPR isolates significantly increased the growth of tea plant in nursery conditions. Thus this study underlines the commercial potential of these selected PGPR isolates for sustainable tea cultivation.

Effects of selected Indonesian plant extracts on E. cuniculi infection in vivo.

The present study was conducted to evaluate the methanolic extracts from several plant leaves widely used in traditional medicine to cure digestive tract disorders and in the self-medication of wild animals such as non-human primates, namely Archidendron fagifolium, Diospyros sumatrana, Shorea sumatrana, and Piper betle leaves, with regard to their antimicrosporidial activity against Encephalitozoon cuniculi in immunocompetent BALB/c mice determined using molecular detection of microsporidial DNA (qPCR) in various tissues and body fluids of infected, treated mice. Of the plant extracts tested, Diospyros sumatrana provided the most promising results, reducing spore shedding by 88% compared to untreated controls. Moreover, total burden per 1 g of tissue in the D. sumatrana extract-treated group reached 87% reduction compared to untreated controls, which was comparable to the effect of the standard drug, Albendazole. This data represents the baseline necessary for further research focused on determining the structure, activity and modes of action of the active compounds, mainly of D. sumatrana, enabling subsequent development of antimicrosporidial remedies.

Behavior of yeast inoculated during semi-dry coffee fermentation and the effect on chemical and sensorial properties of the final beverage.

Pulped Mundo Novo and Ouro Amarelo coffee beans were inoculated with Saccharomyces cerevisiae (CCMA 0200 and CCMA 0543) during semi-dry coffee fermentation and compared with a non-inoculated control. Samples were collected throughout the fermentation process (12days) to evaluate the persistence of the inoculum by Real-Time quantitative PCR (qPCR). Also, the chemical composition of the beans was determined by HPLC and GC-MS and the roasted beans were sensorial evaluated using the cupping test. S. cerevisiae CCMA 0543 had an average population of 5.6logcell/g (Ouro Amarelo cultivar) and 5.5logcell/g (Mundo Novo cultivar). Citric, malic, succinic and acetic acid were found in all samples, along with sucrose, fructose, and glucose. There were 104 volatile compounds detected: 49 and 55 in green and roasted coffee, respectively. All coffee samples scored over 80 points in the cupping test, indicating they were specialty-grade. Inoculation with the CCMA 0543 strain performed better than the CCMA 0200 strain. This is the first time that qPCR has been used to assess the persistence of the inoculated strains populations during coffee processing. Strain CCMA 0543 was the most suitable as an inoculant due to its enhanced persistence during the process and number of volatile compounds produced.

Hemolytic capability and expression of a putative haem oxygenase-encoding gene by blood isolates of Candida tropicalis are influenced by iron deprivation and the presence of hemoglobin and erythrocytes.

Although hemolytic activity is known to be a putative virulence factor contributing to candidal pathogenesis, its production by Candida tropicalis, a species closely related to Candida albicans, is poor understood. The present study was undertaken to evaluate the hemolytic activity and the expression level of a putative haem oxygenase encoding gene by blood isolates of C. tropicalis following growth in iron deprivation, and in the presence of hemoglobin and erythrocytes. The lowest values of hemolytic activity were observed in cell-free culture supernatants of isolates growing in iron-restricted medium (RPMI medium and RPMI medium supplemented with iron chelator bathophenanthrolindisulphonic acid). Hemolysis was increased in the presence of either hemoglobin or erythrocytes. Reverse transcriptase PCR analysis showed that the putative haem oxygenase encoding gene (CtHMX1), potentially related with iron uptake, was up-regulated (p < 0.001) following growth in iron deprivation and in the presence of hemoglobin; CtHMX1 was repressed in the presence of human erythrocytes (p < 0.001). Our data suggest that hemoglobin had positive effect in the production of hemolytic factor and gene expression related to iron uptake in C. tropicalis.

Breakthrough fungemia due to Candida fermentati with fks1p mutation under micafungin treatment in a cord blood transplant recipient.

The prophylactic use of antifungal drugs in allogeneic hematopoietic cell transplant recipients has revealed that the rate of non-albicans candidemia has increased. We herein report the case of a patient with adult T-cell leukemia who developed candidemia due to Candida fermentati during micafungin treatment after cord blood transplantation. The isolate was identified on day 47 by sequencing of the internal transcribed spacer region of the ribosomal RNA gene. The sequencing of the hot spot region of fks1p of isolate revealed naturally occurring amino acid substitutions, which conferred reduced echinocandin susceptibility. This case highlights that breakthrough candidemia due to C. fermentati occurred in a patient receiving micafungin treatment.

Identification of Chinese Caterpillar Medicinal Mushroom, Ophiocordyceps sinensis (Ascomycetes) from Counterfeit Species.

Ophiocordyceps sinensis is a valuable traditional Chinese medicine with a high market price. In this study, a polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) method based on 2 enzymes was developed to distinguish O. sinensis from 6 common counterfeit species. To verify the applicability of this method, we experimentally tested O. sinensis organisms, tablet preparations made from O. sinensis, and cultured mycelia isolated from O. sinensis. To validate the results from this PCR-RFLP method, all real samples were identified by internal transcribed spacer sequencing. This is, to our knowledge, the first time the PCR-RFLP method has been applied to identify O. sinensis. The selection of 2 restrictive enzymes for identification dramatically improved the accuracy and efficiency of this method. It is the great advantage of this method that sampling from either of 2 parts of O. sinensis-the fruiting body or the caterpillar body-would not cause any difference in the final experimental results. Therefore, this method is not only feasible for testing crude drugs of O. sinensis but it is also useful when the crude drugs are broken down into powder or made into tablets, demonstrating the promising prospect of application in quality control.