S-ITS2 rDNA Research Articles

Morphological and molecular identification of Heterakis gallinarum in Gallus gallus and its phylogenetic analysis based on ITS1-5.8S-ITS2 rDNA fragment in Kashmir valley, India.

Heterakis gallinarum is one of the most common round worms infecting wide range of terrestrial birds including domestic fowl. H. dispar and H. isolonche are the species of same genus which infect other hosts predominantly aquatic birds, but show morphological similarities mainly females of these species are difficult to identify. Here, we described the morphologically distinct features of H. gallinarum along with identification through molecular analysis using ITS1-5.8S-ITS2 ribosomal DNA. Four hundred gastrointestinal tracts of domestic fowl were analysed for a period of 2years. Light microscopy and Scanning electron microscopy (SEM), was used for determining the morphometric measurements and characteristic morphological features of H. gallinarum. A PCR based DNA sequencing of ITS1-5.8S-ITS2 ribosomal DNA was used for molecular characterization and phylogenetic analysis was carried out through MEGA 11. Ninety gastrointestinal tracts were found to be infected with Heterakis gallinarum. The male worm displayed well developed lateral and caudal alae, caudal end had an anal sucker, 12 pairs of caudal papillae and cloaca from which 2 unequal spicules emerged. Female had visible vaginal bends, vulva present approximately in the middle of the body, and caudal end had a long pointed tail. Sequence analysis revealed that the PCR products of ITS1-5.8S-ITS2 rDNA were 600bp in size and showed 96-98% similarity with the ITS1-5.8S-ITS2 sequences of H. gallinarum on BLAST analysis. Our ITS sequence of H. gallinarum showed a nucleotide divergence of 4.9% with H. gallinarum of Lucknow India. The phylogenetic analysis clearly differentiated H. gallinarum from the other representatives of the genus and revealed the relationship among different Heterakis species. The study revealed the infection status of Heterakis gallinarum prevailing among domestic fowl, and need of strategic control measures. The results of the study suggest that ITS rDNA would serve as a potential molecular marker in identification, diagnosis and systematics of Heterakis species.

New species and new records of Laccaria (Agaricales, Basidiomycota) from Northern Thailand.

Two new species Laccariapseudoalba and L.subroseoalbescens are described and illustrated, based on morphological characteristics and molecular phylogenetic analysis. Two new records, Laccariaumbilicata and L.yunnanensis from Thailand, are also reported. Laccariasubroseoalbescens is characterized by small basidiomata, stipe equal with an enlarged base, and nearly subclavate, pale pink to light orange. Laccariapseudoalba is characterized by pale orange to orange white pileus, has umbo when young on the pileus, and fistulose stipe of the pale to pastel red color. Phylogenetic analysis based on sequence data from rDNA internal transcribed spacer ITS1-5.8S-ITS2 rDNA (ITS), nuc 28S rDNA (28S), RNA polymerase II subunit 2 (rpb2), and translation elongation factor 1-α (tef1-α) are provided as further evidence. Molecular analysis confirms the phylogenetic positions of the two new species and two new records. The differences in characteristics of these two new species and closely related species are discussed herein.

Identification of a new species of Gyrodactylus von Nordmann, 1832 (Monogenoidea Gyrodactylidae) isolated from Diptychus maculatus in Yarkand River, Xinjiang, China

To investigate Gyrodactylus infection of fish in the river system of Xinjiang (China), Gyrodactylus individuals were isolated from specimens of Diptychus maculatus. Morphological characterization and phylogenetic analysis based on ITS1-5.8S-ITS2 rDNA locus revealed that the gyrodactylids belong to new species. Gyrodactylus diptychi n. sp. differs significantly in the morphology of the haptoral structures from 12 known species of Gyrodactylus found in fishes of the subfamily Schizothoracinae. In particular, G. diptychi n. sp. has a relatively short dorsal bar with thick and large ends, flat and straight hamuli roots, and small ventral bar processes. Furthermore, G. diptychi n. sp. is the only representative of Gyrodactylus found on D. maculatus. Using the BLASTn search of ITS1-5.8S-ITS2 rDNA sequences in GenBank and the Bayesian Information and Maximum Likelihood methods, we constructed phylogenetic trees for G. diptychi n. sp. As a result, our studies clearly identified that G. diptychi n. sp. was the first Gyrodactylus monogenean isolated from D. maculatus and a new species belonged to the subgenus Limnonephrotus.

Morphological and molecular analyses reveal two new species of Grifola (Polyporales) from Yunnan, China.

Species of Grifola are famous edible mushrooms and are deeply loved by consumers around the world. Most species of this genus have been described and recorded in Oceania, Europe and South America, with only Grifolafrondosa being recorded in Asia. In this study, two novel species of Grifola from southwestern China (Asia) are introduced. Macro and micromorphological characters are described. Grifolaedulissp. nov. present medium-size basidiomata with gray to gray-brown lobes upper surface, mostly tibiiform or narrowly clavate, rarely narrowly lageniform or ellipsoid chlamydospores, cuticle hyphae terminal segments slightly enlarged. Grifolasinensissp. nov. has white to grayish white lobes upper surface, mostly ellipsoid, rarely narrowly utriform chlamydospores, and broadly ellipsoid to ellipsoid basidiospores (4.6-7.9 × 3.0-5.9 μm). The two new species are supported by phylogenetic analyses of combined nuclear rDNA internal transcribed spacer ITS1-5.8S-ITS2 rDNA (ITS) and β-tubulin (TUBB). Moreover, the genetic distance between TUBB sequences of those specimen from GenBank was 1.76-1.9%. Thus, the conspecificity relationship of our specimens remains uncertain, and further specimens are required to conclusively confirm its identity.

Surface Ultrastructure and Molecular Studies of Clinostomum complanatum (Rudolphi, 1814) Braun, 1899 (Trematoda: Clinostomidae) Metacercariae in some Freshwater Fishes from Sulaimani Province, Iraq

Clinostomum is a trematode genus in the family Clinostomidae. The mentioned trematode species is parasitizing many fish species as intermediate hosts, while piscivorous birds and mammals are the main definitive hosts. Total of 58 metacercariae of Clinostomum larvae were dissected out from 25 infected fish specimens from eight different species from a number of water bodies of Sulaimani Province, Kurdistan Region, Iraq. In this investigation, 959 fish specimens were collected. These includes five cyprinid species: Capoeta trutta (Heckel), C. umbla (Heckel), Carasobarbus kosswigi (Ladiges), Cyprinion macrostomum Heckel and Garra rufa (Heckel), two leuciscid species: Alburnus sellal Heckel and Squalius lepidus Heckel and the mugilid fish Planiliza abu (Heckel). The prevalence of infection for each of these species was 1.8%, 5%, 20%, 1.5%, 1.7%, 4.9%, 4.9% and 1.3%, respectively. The morphology of the Clinostomum metacercariae was studied by using a compound microscope; their ultra-morphology was evaluated with a scanning electron microscopy (SEM); and the molecular analysis were performed by amplifying, sequencing and comparing the ITS1-5.8S-ITS2 and 28S rDNA gene loci from isolated Clinostomum metacercariae. The obtained sequences confirmed that all metacercariae of the Clinostomum, collected during the present study, represented with C. complanatum (Rudolphi, 1814) based on percent identity with sequences in the GenBank subject database. The molecular characterisations of the C. complanatum metacercariae in the present study were deposited in GenBank NCBI.

Characterization of three-nucleate Rhizoctonia AG-E based on their morphology and phylogeny

The genus Rhizoctonia has been classified into two main groups according to the number of nuclei. Binucleate Rhizoctonia strains have two nuclei in each cell, whereas multinucleate Rhizoctonia fungi were observed to have a variable number of nuclei ranging from 4 to 16 in each cell. In the study, twelve Polish isolates were tested. According to ITS1-5,8S-ITS2 rDNA sequences, the isolates were classified in the AG-E. Their affiliation to AG was confirmed by anastomosis reactions with tester isolates. The number of nuclei was counted with DAPI staining under a fluorescent microscope, and the diameter of the hyphae was also measured. Not all AG-E isolates had the same number of nuclei in their cells: one group among these fungi produced cells with a diverse number of nuclei, usually 3; however, this number ranged from 2 to 4, making the average number of nuclei close to 3. It can be assumed that all isolates with three nuclei belong to this group, which may greatly facilitate the preliminary identification of trinucleate isolates of Rhizoctonia spp. belonging to AG-E. Based on these characters, we call these isolates AG-E-3n isolates. The thiamine requirement is not helpful in classifying and describing the AG-E strains.

New species of Gyrodactylus von Nordmann, 1832 (Monogenoidea: Gyrodactylidae) from Gymnodiptychus dybowskii (Kessler, 1874) (Schizothoracinae) in the Kunes River (Yili River basin), China

Yili River system hosts a diverse fauna of fishes and parasites. Gymnodiptychus dybowskii is a rare and endangered aboriginal cold-water fish inhabit in the Yili river system. Our research identified a new species Gyrodactylus gymnodiptychi n. sp. isolated from G. dybowskii in the Kunes River (Yili River, China). Morphological comparison revealed identifiable differences between the new species and other parasites, including Gyrodactylus aksuensis, and Gyrodactylus tokobaevi, which are two known parasites living in G. dybowskii inhabit in the Aksu River west of Frunze (Kyrgyzstan), as well as Gyrodactylus montanus living in Shizothorax intermedius inhabited in the Tadzhikistan or Uzbekistan. Especially, the dorsal bar of G. gymnodiptychi n. sp. was raised at both ends with a hollow, and its hamulus roots were curved inward. The BLASTN search of GenBank did not detect any other ITS1-5.8S-ITS2 rDNA sequences same as G. gymnodiptychi's. Using the Bayesian Information and Maximum Likelihood methods to analyze the ITS1-5.8S-ITS2 rDNA gene sequences, we constructed phylogenetic trees for G. gymnodiptychi n. sp. Accordingly, our morphological and molecular research indicated that G. gymnodiptychi n. sp. was not only a new species of parasites but also the first Gyrodactylus member identified in the Yili River in China.

Three new species and a new record of Cortinarius section Camphorati from southwestern China

ABSTRACT Cortinarius sect. Camphorati consists of telamonioid species with violet basidioma, strong odor, and distinct cheilocystidia. In this study, phylogenetic analysis based on nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS) and partial nuc rDNA 28S sequences has revealed three new species of the section from southwestern China, namely, C. longicystidiatus, C. megacystidiatus, and C. paraputorius, and a newly recorded species from southwestern China, viz., C. camphoratus, supplemented by the support from calculations of genetic distances of ITS sequences. Detailed descriptions of the four species are given with photographs, and their micromorphological characteristics are statistically compared. For species delimitation within the section, the size of basidiospores, the morphology of cheilocystidia, and the associated vegetation types and tree species are informative. A key to species in section Camphorati from the Northern Hemisphere is provided, and their geographic distributions and ecology are discussed.

Comparison of Fungal Genera Isolated from Cucumber Plants and Rhizosphere Soil by Using Various Cultural Media.

Plant endophytic fungi and rhizosphere soil fungi are often reported as biocontrol agents against plant pathogens or with plant growth promotion potential. Four treatments were performed in field and greenhouse experiments where cucumber plants were inoculated with Trichoderma harzianum and Fusarium oxysporum in 2022. The roots, stems and leaves of cucumber plants and their rhizosphere soil were collected twice individually from the field and greenhouse for isolation of cucumber endophytic and rhizosphere soil fungi. All fungal strains were identified through sequence similarity of the ITS1-5.8s-ITS2 rDNA region. The potato dextrose agar (PDA) media yielded the highest number of genera isolated from cucumber plants, rhizosphere soil and both compared to other media. There were no significant differences among the four media for the isolation of all cucumber endophytic fungi. However, in the roots, the number of endophytic fungi isolated by MRBA was significantly higher than that isolated on malt extract agar (MEA), while in the stems, the number of fungi isolated with PDA was significantly higher than that isolated with Martin's rose bengal agar medium (MRBA). PDA had significantly higher isolation efficiency for the rhizosphere soil fungi than MRBA. The 28 fungal genera had high isolation efficiency, and the endophytic Trichoderma strains were significantly more isolated by MEA than those of MRBA. It is suggested that PDA can be used as a basic medium, and different cultural media can be considered for specific fungal genera.

First Report of Rhizopus stolonifer Causing Premature Soft Rot of Jackfruit in Bangladesh.

The jackfruit tree (Artocarpus heterophyllus) is native to South and South-east Asia including Bangladesh. It is a commercially important tropical tree species that produces fruit, food, fodder, and high-quality wood (Gupta et al. 2022). During surveys in February 2022, soft rot on immature fruit at approximately 70% incidence was observed in several plantations and homesteads in the Sylhet district of Bangladesh. Infected fruit had black patches surrounded by wide bands of white, powdery masses. The patches enlarged with fruit maturation, and in some cases, covered the entire fruit. Symptomatic fruit were collected, surface sterilized with 70% ethanol for 1 min, and washed 3 times with sterile distilled water. Fen air-dried, and small pieces from the margins of lesions were transferred to potato dextrose agar (PDA). The plates were incubated at 25°C in the dark. Two-day-old colonies had diffuse, gray cottony mycelia that were hyaline and aseptate under the microscope. Sporangiophores measuring 0.6-2.5mm in length and 18 to 23μm in diameter had rhizoids and stolons at their bases. Sporangia were almost spherical and were 125μm (±65μm, n=50) in diameter. Sporangiospores were ellipsoid to ovoid and measured 3.5 to 9.32μm × 2.82 to 5.86μm (x̄= 5.86×4.1μm, n=50). Based on these morphological features, the isolates were identified preliminarily as Rhizopus stolonifer (García-Estrada et al. 2019; Lin et al. 2017). To identify the pathogen molecularly, genomic DNA was extracted using the FavorPrep Fungi/Yeast Genomic DNA extraction Mini Kit (Taiwan). Polymerase chain reaction (PCR) amplification of ITS1-5.8S-ITS2 rDNA was done using primers ITS4 and ITS5 (White et al. 1990) following the procedure of Khan and Bhadauria (2019). The PCR product was then sequenced by Macrogen, Korea. A BLAST search in GenBank revealed that isolate JR02 (GenBank accession OP692731) was 100% identical to R. stolonifer (GenBank accession MT256940). In pathogenicity tests,10 healthy young fruit at a similar maturity stage as the ones found diseased were collected from a orchard where the disease was not observed. Fruit were surface sterilized with 70% ethylalcohol and washed with sterile distilled water. Wounded (using a sterilized needle) and non-wounded fruits were inoculated with 20µl of a spore suspension (1×106/ml). Sterile distilled water was used for the controls. Inoculated fruit were covered with sterile cloth, transferred to perforated plastic bags with moistened blotting paper, and incubated at 25°C in the dark. Symptoms were first observed after 2 days on wounded fruit, but no symptoms developed on controls and non-wounded fruit. Rhizopus stolonifer was re-isolated from infected fruit, thus fulfilling Koch's postulates. Rhizopus rot is a devastating disease causing premature fruit drop, reduced crop yield, and post-harvest rot of jackfruit and other fruits and vegetables (Sabtu et al. 2019). Three Rhizopus species namely R. stolonifer, R. artocarpi and R. oryzae have been reported causing fruit rot of jackfruit in the tropics including Mexico, India and Hawaii (García-Estrada et al., 2019; Babu et al., 2018; Nelson, 2005). Appropriate management strategies are needed to be developed to prevent premature rot of jackfruit. To our knowledge, this is the first report of R. stolonifer causing premature soft rot of jackfruit in Bangladesh.

Molecular characterisation of three species of Coitocaecum (Digenea: Opecoelidae) infecting Clinus superciliosus (Clinidae) in South Africa, with description of Coitocaecum brayi sp. n.

The genus Coitocaecum Nicoll, 1915 is part of the most speciose digenean family, the Opecoelidae Ozaki, 1925, which is found globally in both freshwater and marine fishes. Fifteen opecoelid species have been reported from marine fishes in South Africa, yet only one species of Coitocaecum has been described from this region: Coitocaecum capense Bray, 1987. During an explorative study of the digeneans of the endemic, intertidal fish Clinus superciliosus (Linnaeus) from the Saldanha Bay area, Cape Town harbour, Hermanus, the Tsitsikamma section of the Garden Route National Park and Chintsa East in South Africa, a total of three distinct species of Coitocaecum were identified based on morphological and molecular (28S rDNA, ITS1-5.8S-ITS2 rDNA and COI mtDNA) data: the previously mentioned C. capense, Coitocaecum brayi sp. n. and a third, unnamed species. We provide the first molecular characterisation of species of Coitocaecum from South Africa, accompanied by detailed morphological descriptions. This study illustrates the importance of an integrated taxonomic approach, especially when studying species with similar morphology. These findings further emphasise the lack of information on the true diversity and molecular data for trematodes of marine fishes in South Africa, creating a great capacity for future explorative taxonomic studies and highlighting the use of intertidal areas for conducting such research.

Crude Enzyme Concentrate of Filamentous Fungus Hydrolyzed Chitosan to Obtain Oligomers of Different Sizes.

Chitosan is a non-cytotoxic polysaccharide that, upon hydrolysis, releases oligomers of different sizes that may have antioxidant, antimicrobial activity and the inhibition of cancer cell growth, among other applications. It is, therefore, a hydrolysis process with great biotechnological relevance. Thus, this study aims to use a crude enzyme concentrate (CEC) produced by a filamentous fungus to obtain oligomers with different molecular weights. The microorganism was cultivated in a liquid medium (modified Czapeck-with carboxymethylcellulose as enzyme inducer). The enzymes present in the CEC were identified by LC-MS/MS, with an emphasis on cellobiohydrolase (E.C 3.2.1.91). The fungus of the Aspergillus genus was identified by amplifying the ITS1-5.8S-ITS2 rDNA region and metaproteomic analysis, where the excreted enzymes were identified with sequence coverage greater than 84% to A. nidulans. Chitosan hydrolysis assays compared the CEC with the commercial enzyme (Celluclast 1.5 L®). The ability to reduce the initial molecular mass of chitosan by 47.80, 75.24, and 93.26% after 2.0, 5.0, and 24 h of reaction, respectively, was observed. FTIR analyses revealed lower absorbance of chitosan oligomers' spectral signals, and their crystallinity was reduced after 3 h of hydrolysis. Based on these results, we can conclude that the crude enzyme concentrate showed a significant technological potential for obtaining chitosan oligomers of different sizes.

Morphological and molecular characterization of three new species of Gyrodactylus (Monogenea: Gyrodactylidae) infecting Sicydium salvini (Teleostei: Gobiidae) in Mexican rivers draining into the Pacific Ocean.

The genus Gyrodactylus von Nordmann, 1832 is one of the most diverse within the class Monogenea; it contains mainly parasites of freshwater and marine teleost fishes. Around 40 species of Gyrodactylus have been described from gobiid fishes; mainly in Europe, as only two species are known from the Americas. In this study, we describe three new gyrodactylids from the body surface and fins of the goby Sicydium salvini (Gobiidae, Sycydiinae), which has a wide distribution on the Pacific coast, from Mexico to Panama. We describe Gyrodactylus oaxacae n. sp., G. atoyacensis n. sp. and G. salvini n. sp. collected from rivers draining to the eastern Pacific in the state of Oaxaca, Mexico. Morphologically, G. atoyacensis n. sp. and G. salvini n. sp. are very similar, and both are easily differentiated from G. oaxacae n. sp. Phylogenetic hypotheses based on sequences of the Internal Transcribed Spacers (ITS1-5.8S-ITS2 rDNA) and the D2+D3 domains of the large ribosomal subunit (28S rDNA) support the erection of the three new taxa; and suggest that G. atoyacensis n. sp. and G. salvini n. sp. are sister species. These gyrodactylids are the first monogeneans described from gobies of the genus Sicydium in Mexico.

Detection of Naganishia albida Yeasts in Dermatological Patients.

The yeasts Cryptococcus albidus (Naganishia albida) usually occur on natural substrates and rarely are the etiological factor of different mycoses. More than a half of mycosis cases described in the literature were reported during the period from 2004 to 2021. In this regard, evaluation of yeast sensitivity to antimycotic drugs is as important as their identification. In the present study, two yeast isolates from the skin of female patients (age 7 and 74 years) with infective dermatitis (ICD-10-CM Code L30.3) were studied. Common identification of the isolates, MALDI-TOF mass spectrometry, and analysis of the nucleotide sequences of the ITS1-5.8S-ITS2 rDNA region showed that they belong to the species N. albida. The sensitivity of the obtained strains to antimycotics of three different chemical groups, namely itraconazole, naftifine, and amphotericin B, determined by microdilution method in a synthetic medium showed the following minimum inhibitory concentrations: 64-128, 16, and 0.125-4 μg/ml, respectively. It was found that the sensitivity of this yeast to pooled human serum was 30-47%, i.e. lower by 1.9-2.9 times than the sensitivity of the collection strains of C. albicans and C. neoformans. This result could be explained by lower prevalence of N. albida in the human population in comparison with these species. However, the sensitivity of N. albida strains to the low-molecular-weight fraction of serum was approximately the same as in C. albicans and C. neoformans, which indicates their high sensitivity to antimicrobial peptides.

Morphological and molecular analyses reveal two new species of Termitomyces (Agaricales, Lyophyllaceae) and morphological variability of T.intermedius.

Two new species, Termitomycestigrinus and T.yunnanensis are described based on specimens collected from southwestern China. Termitomycesyunnanensis is morphologically characterized by a conspicuously venose pileus surface that is grey, olive grey, light grey to greenish grey at center, light grey towards margin, and a cylindrical white stipe. Termitomycestigrinus is morphologically characterized by a densely tomentose to tomentose-squamulose pileus showing alternating greyish white and dark grey zones, and a stipe that is bulbous at the base. The two new species are supported by phylogenetic analyses of combined nuclear rDNA internal transcribed spacer ITS1-5.8S-ITS2 rDNA (ITS), the mitochondrial rDNA small subunit (mrSSU) and the nuclear rDNA large subunit (nrLSU). The morphological variability of T.intermedius, including five specimens newly collected from Yunnan Province, China, is also discussed. The collections showed variability in colour of the stipe surface and in the shape of cheilocystidia when compared to the original description. Full descriptions of the two new species and of T.intermedius, as well as a taxonomic key to the 14 Termitomyces species reported from China are provided.

Prey-mimetism in cercariae of Apatemon (Digenea, Strigeidae) in freshwater in northern latitudes.

Cercariae, the free-living larval stages of trematodes, have adopted an amazing variety of transmission strategies. One of them is prey-mimetism, i.e. cercariae mimicking prey to attract motile hosts to be eaten. In a period between 2002 and 2019, we examined small planorbid snails, Bathyomphalus contortus, Gyraulus parvus and Planorbis planorbis from lakes in Finland and Iceland and from the Curonian Lagoon in Lithuania. Cercariae with conspicuously enlarged tails and unusual swimming behaviour, likely mimicking invertebrate prey, were detected and studied by the use of morphological and molecular (cox1, ITS1-5.8S-ITS2 and 28S rDNA) methods. Cercariae of two species belonging to the genus Apatemon (Strigeidae) were recognised. We consider Apatemon sp. 5 ex P. planorbis from the Curonian Lagoon identical to Cercaria globocaudata U. Szidat, 1940. Cercariae ex G. parvus from Iceland and ex B. contortus from Finland were conspecific, and we named them Apatemon sp. 6; these cercariae could not be associated with any known species. For the first time, we verified that cercariae of the Bulbocauda group belong to the genus Apatemon. We provide a mini-review on records of furcocercariae of the family Strigeidae with enlarged tails reported in freshwaters of the northern hemisphere and reveal that it is not only Apatemon but also Australapatemon and most likely Strigea which belong to the Bulbocauda group, rendering it a purely ecological assemblage. Understanding which invertebrate swimming behaviour these cercariae are mimicking will enhance our knowledge of the processes behind trematode transmission and will help to assess evolutionary pathways of host-finding strategies in trematodes.

Molecular identification of yeasts from Turkish traditional cheeses: Extracellular enzyme activities and physiological properties important for dairy industry

Abstract. Gunay M, Genc TT. 2023. Molecular identification of yeasts from Turkish traditional cheeses: Extracellular enzyme activities and physiological properties important for dairy industry. Nusantara Bioscience 15: 1-11. The determination of yeast microbiota in cheeses and the physiological properties of yeasts are very important for the dairy industry. In addition, the physiological features, proteolytic and lipolytic activities, and stress tolerance of yeasts have a significant role in the selection of starter yeast species for cheese ripening. This study aimed to determine industrially important yeasts isolated from cheese samples. Molecular techniques identified the isolated yeast strains. The yeast strains’ extracellular enzyme activities, fermentation capacities, and thermotolerance and osmotolerance properties were also evaluated. A total of 81 yeast strains were isolated and characterized from three types of cheese samples. PCR-RFLP determined the isolated yeast strains and sequence analysis of ITS1-5.8S-ITS2 and 26S rDNA regions. A maximum parsimony tree was constructed by MEGA X software to evaluate the phylogenetic relationship of identified yeast strains. Candida intermedia, Candida parapsilosis, Clavispora lusitaniae, Debaryomyces hansenii, Kluyveromyces marxianus, Pichia kudriavzevii, and Wickerhamomyces anomalus yeast species were identified on cheese samples. The distribution of identified yeast species on cheese samples was determined as 48.1% for W. anomalus, 17.3% for K. marxianus, 14.8% for C. parapsilosis, 8.6% for D. hansenii, 4.9% for Cl. lusitaniae, 3.7% for C. intermedia and 2.5% for P. kudriavzevii. The W. anomalus yeast species was common in three cheese types. All strains of W. anomalus and P. kudriavzevii yeast species, three C. parapsilosis, and two Cl. lusitaniae yeast strains have important physiological properties for industrial applications. These yeast strains have the potential to be used in combination as starter cultures to improve cheese maturation in the future. This comprehensive study identifies yeast species by ITS1-5.8S-ITS2 and 26S rDNA regions and determines industrially important yeast species using multiple criteria (extracellular enzyme activity, stress tolerance, and fermentation capacity).

Molecular Identification of Yeast Strains with High Fermentation Capacity Isolated from Kırkağaç Melon Varieties

The yield of fermentation process, bioethanol, is one of the important product obtained from renewable energy sources. Yeasts are commonly used in ethanol production due to their high ethanol tolerance, high ethanol productivity, and their ability to ferment different kinds of simple sugars. Saccharomyces cerevisiae yeast species is the most commonly used in industrial ethanol production. Therefore, isolation and identification of new yeast strains with high fermentation capacity is very important for the bioethanol industry. The aim of this study was isolation of novel and industrially applicable yeast species that can be used in bioethanol production. Yeast strains were isolated from the Dalaman, Altınbaş and Kıvırcık sarı dilim melon varieties that are unique to Kırkağaç district of Manisa province. The fermentation ability of yeast strains was evaluated and the selected yeast strains were identified by PCR-RFLP analysis of 26S and ITS1-5.8S-ITS2 rDNA gene regions. Sequence analysis of the 26S rDNA-D1/D2 gene region was used for molecular identification of yeast strains. The phylogenetic analysis of identified yeast strains was carried out using the MEGA XI phylogenetic analysis tool. It was determined that 14 of the 53 yeast strains isolated were able to ferment glucose and sucrose, and four of them were able to ferment lactose. The yeast strains having the fermentation capacity were identified as Pichia kluyverii, Lachancea thermotolerans, Pichia kudriavzevii, Saccharomyces cf. cerevisiae/paradoxus and Saccharomyces cerevisiae. These yeast strains have the potential to be used in the bioethanol industry after other physiological and biochemical tests are complete

Not all bad: Gyromitrin has a limited distribution in the false morels as determined by a new ultra high-performance liquid chromatography method

ABSTRACT Gyromitrin (acetaldehyde N-methyl-N-formylhydrazone) and its homologs are deadly mycotoxins produced most infamously by the lorchel (also known as false morel) Gyromitra esculenta, which is paradoxically consumed as a delicacy in some parts of the world. There is much speculation about the presence of gyromitrin in other species of the lorchel family (Discinaceae), but no studies have broadly assessed its distribution. Given the history of poisonings associated with the consumption of G. esculenta and G. ambigua, we hypothesized that gyromitrin evolved in the last common ancestor of these taxa and would be present in their descendants with adaptive loss of function in the nested truffle clade, Hydnotrya. To test this hypothesis, we developed a sensitive analytical derivatization method for the detection of gyromitrin using 2,4-dinitrobenzaldehyde as the derivatization reagent. In total, we analyzed 66 specimens for the presence of gyromitrin over 105 tests. Moreover, we sequenced the nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS barcode) and nuc 28S rDNA to assist in species identification and to infer a supporting phylogenetic tree. We detected gyromitrin in all tested specimens from the G. esculenta group as well as G. leucoxantha. This distribution is consistent with a model of rapid evolution coupled with horizontal transfer, which is typical for secondary metabolites. We clarified that gyromitrin production in Discinaceae is both discontinuous and more limited than previously thought. Further research is required to elucidate the gyromitrin biosynthesis gene cluster and its evolutionary history in lorchels.

Molecular and Physiological Diversity of Indigenous Yeasts Isolated from Spontaneously Fermented Wine Wort from Ilfov County, Romania.

(1) Background: Wine yeast research offers the possibility of isolating new strains with distinct metabolic properties due to the geographical location of the vineyard and the processes used in winemaking. Our study deals with the isolation and identification of six yeasts from spontaneously fermented wine wort from Romania and their characterization as new potential starter culture for traditional beverages, for food industry or biomedicine. (2) Materials and methods: The isolates were identified using conventional taxonomy tests, phenotypic phylogeny analysis (Biolog YT), MALDI-TOF mass spectrometry, PCR-RFLP, and sequencing of the ITS1-5,8S-ITS2 rDNA region. The capacity of the yeasts to grow under thermal, ionic, and osmotic stress was determined. The safe status was confirmed by testing virulence and pathogenicity factors. Assays were performed in order to evaluate the growth inhibition of Candida strains and determine the antimicrobial mechanism of action. (3) Results and discussions: The yeast isolates were identified as belonging to the Metschinikowia, Hanseniaspora, Torulaspora, Pichia, and Saccharomyces genera. All the isolates were able to develop under the tested stress conditions and were confirmed as safe. With the exception of S. cerevisiae CMGB-MS1-1, all the isolates showed good antimicrobial activity based on competition for iron ions or production of killer toxins. (4) Conclusions: The results revealed the resistance of our yeasts to environmental conditions related to industrial and biomedical applications and their high potential as starter cultures and biocontrol agents, respectively.