Inocybe brijunica sp. nov., a new ectomycorrhizal fungus from Mediterranean Croatia revealed by morphology and multilocus phylogenetic analysis

A new ectomycorrhizal species was discovered during the first survey of fungal diversity at Brijuni National Park (Croatia), which consists of 14 islands and islets. The National Park is located in the Mediterranean Biogeographical Region, a prominent climate change hot-spot. Inocybe brijunica sp. nov., from sect. Hysterices (Agaricales, Inocybaceae), is described based on morphology and multilocus phylogenetic data. The holotype collection was found at the edge between grassland and Quercus ilex forest with a few planted Pinus pinea trees, on Veli Brijun Island, the largest island of the archipelago. It is easily recognized by a conspicuous orange to orange–red–brown membranaceous surface layer located at or just above the basal part of the stipe. Other distinctive features of I. brijunica are the medium brown, radially fibrillose to rimose pileus; pale to medium brown stipe with fugacious cortina; relatively small, amygdaliform to phaseoliform, and smooth basidiospores, measuring ca. 6.5–9 × 4–5.5 µm; thick-walled, utriform, lageniform or fusiform pleurocystidia (lamprocystidia) with crystals and mostly not yellowing in alkaline solutions; cheilocystidia of two types (lamprocystidia and leptocystidia); and the presence of abundant caulocystidia only in the upper 2–3 mm of the stipe. Phylogenetic reconstruction of a concatenated dataset of the internal transcribed spacer region (ITS), the nuclear 28S rRNA gene (nrLSU), and the second largest subunit of RNA polymerase II (rpb2) resolved I. brijunica and I. glabripes as sister species

Coprinus leucostictus rediscovered after a century, epitypified, and its generic position in Hausknechtia resolved by multigene phylogenetic analysis of Psathyrellaceae

About a century after the first finding in northern Vietnam (1908), Coprinus leucostictus is rediscovered on 12 localities in southern India and southern to southeastern China, growing in evergreen subtropical or tropical forests. It is morphologically a rather unique species with coprinoid basidiomata, strongly branched and diverticulate veil hyphae, and a hymeniderm pileipellis. The BLAST search of ITS and tef-1a sequences showed its close relationship to Hausknechtia floriformis, which is not clear based on morphological characters. Multigene phylogenetic analysis of a concatenated dataset of ITS, LSU, tef-1a, and -tubulin sequences, revealed C. leucostictus and H. floriformis as separate, but sister species. Molecular phylogenetic relationships within the family Psathyrellaceae (including 17 genera) are presented in the phylogram. The genera Hausknechtia and Candolleomyces formed two well-supported lineages and were recovered as a monophyletic group. A total of 27 sequences from the genus Hausknechtia were newly generated in this study. Coprinus leucostictus is combined as Hausknechtia leucosticta, its epitype is designated, and the hitherto monotypic genus Hausknechtia is emended. A detailed morphological description of H. leucosticta supplemented with colour photographs and line drawings is provided

CONTRIBUTION OF DNA BARCODING TO THE RESEARCH OF CROATIAN FUNGAL BIODIVERSITY

Cilj doktorske disertacije bio je rješavanje znanstvenih problema u sistematici gljiva metodama integrativne taksonomije koja objedinjuje evaluaciju molekularnih, morfoloških i ekoloških obilježja gljivljih organizama. Poseban naglasak stavljen je na vrednovanje znanstvenog doprinosa DNA barkodiranja u otkrivanju, razgraničenju i identifikaciji srodnih vrsta u različitim skupinama nadzemnih gljiva iz odjeljaka Ascomycota i Basidiomycota. Prikazan je znanstveni opis tri vrste nove za znanost s područja Republike Hrvatske: Inocybe brijunica, I. istriaca i Parasola papillatospora. Kao važan biogeografski doprinos ovoga rada na području Hrvatske po prvi je puta pronađeno 10 vrsta gljiva: Coprinopsis alnivora, Entonaema cinnabarinum, Parasola auricoma, P. crataegi, P. cuniculorum, P. kuehneri, P. malakandensis, P. megasperma, P. nudiceps i P. plicatilis-similis. Vrste C. alnivora i P. malakandensis ujedno su po prvi puta zabilježene i na području Europe. Integrativna taksonomska analiza roda Entonaema rezultirala je određivanjem njegovog filogenetskog i taksonomskog položaja u porodici Hypoxylaceae te izradom identifikacijskog ključa za vrste na svjetskoj razini.The aim of this dissertation was to solve scientific problems in the systematics of fungi using integrative taxonomy, which combines molecular, morphological, and ecological characteristics of fungi. Special emphasis was placed on the contribution of DNA barcoding in the discovery, delimitation, and identification of related species in different groups of epigeous fungi (Ascomycota and Basidiomycota). The scientific description of three species new to science from Croatia was presented: Inocybe brijunica, I. istriaca, and Parasola papillatospora. An important biogeographical contribution was ten species of fungi found for the first time in Croatia: Coprinopsis alnivora, Entonaema cinnabarinum and 8 species of Parasola. Coprinopsis alnivora and P. malakandensis were also recorded for the first time in Europe. The integrative taxonomic analysis of the genus Entonaema led to the determination of their phylogenetic and taxonomic position in the family Hypoxylaceae and the establishment of an identification key of worldwide species within this genus

Selection of Lactobacillus and Enterococcus strain from dry sausages for their potential use as probiotics

Cilj ovog diplomskog rada bio je utvrditi probiotički potencijal vrsta Enterococcus durans (n = 4) i Lactobacillus sakei (n = 9) izoliranih iz tradicionalnih trajnih kobasica. Ispitan je sigurnosni status selektiranih sojeva, kao i njihova sposobnost autoagregacije i preživljavanja u simuliranim gastrointestinalnim uvjetima. Kod niti jednog od selektiranih izolata nije utvrđena rezistencija na korištene antibiotike i hemolitička aktivnost te nije detektirana prisutnost gena koji kodiraju za virulenciju ili produkciju biogenih amina. Svi selektirani sojevi pokazali su lošu sposobnost autoagregacije s prosječnom vrijednosti od 17,67 % nakon 5 h inkubacije. Najveći broj izolata preživio je u uvjetima usne šupljine, s prosječnom vrijednosti od 83,25 % dok je mogućnost preživljavanja značajno smanjena u simuliranim uvjetima želuca. Simulirani uvjeti dvanaesnika pokazali su se nepovoljnima za preživljenje većine izolata, s prosječnim vrijednostima preživljavanja od 0,75 % za sojeve L. sakei te 9,95 % za sojeve E. durans. Usprkos tomu, jedan soj E. durans (Ed3) pokazao je značajno više vrijednosti preživljavanja i potencijalni je kandidat za daljnja istraživanja vezana uz probiotičku primjenu.The aim of this master thesis was to evaluate probiotic potential of Enterococcus durans (n = 4) and Lactobacillus sakei (n = 9) isolated from Croatian traditional dry fermented sausages. Safty of the selected genotypes was investigated, as their ability go autoaggregate and survive under simulated gastrointestinal conditions. None of the selected genotypes showed resistance to tested antibiotics or hemolytic activity and the presence of the virulence determinants or genes encoding for production of biogenic amines were confirmed. All of the selected genotypes exhibited low ability to autoaggregate, with average value of 17.67 after 5 h of incubation. Regarding survival rates in simulated gastrointestinal conditions, all genotypes were best able to tolerate the conditions of oral cavity with average survival rate of 83.25 %. In the simulated environment of the stomach, the viability of bacteria significantly decreased, but the most detrimental effect had simulated conditions of small intestine with average values of 0.75 % for L. sakei strains and 9.95 % for E. durans. However, one E. durans genotype (Ed3) stands out with higher survival rates and can be considered for further analysis as a potential probiotic culture

CONTRIBUTION OF DNA BARCODING TO THE RESEARCH OF CROATIAN FUNGAL BIODIVERSITY

Cilj doktorske disertacije bio je rješavanje znanstvenih problema u sistematici gljiva metodama integrativne taksonomije koja objedinjuje evaluaciju molekularnih, morfoloških i ekoloških obilježja gljivljih organizama. Poseban naglasak stavljen je na vrednovanje znanstvenog doprinosa DNA barkodiranja u otkrivanju, razgraničenju i identifikaciji srodnih vrsta u različitim skupinama nadzemnih gljiva iz odjeljaka Ascomycota i Basidiomycota. Prikazan je znanstveni opis tri vrste nove za znanost s područja Republike Hrvatske: Inocybe brijunica, I. istriaca i Parasola papillatospora. Kao važan biogeografski doprinos ovoga rada na području Hrvatske po prvi je puta pronađeno 10 vrsta gljiva: Coprinopsis alnivora, Entonaema cinnabarinum, Parasola auricoma, P. crataegi, P. cuniculorum, P. kuehneri, P. malakandensis, P. megasperma, P. nudiceps i P. plicatilis-similis. Vrste C. alnivora i P. malakandensis ujedno su po prvi puta zabilježene i na području Europe. Integrativna taksonomska analiza roda Entonaema rezultirala je određivanjem njegovog filogenetskog i taksonomskog položaja u porodici Hypoxylaceae te izradom identifikacijskog ključa za vrste na svjetskoj razini.The aim of this dissertation was to solve scientific problems in the systematics of fungi using integrative taxonomy, which combines molecular, morphological, and ecological characteristics of fungi. Special emphasis was placed on the contribution of DNA barcoding in the discovery, delimitation, and identification of related species in different groups of epigeous fungi (Ascomycota and Basidiomycota). The scientific description of three species new to science from Croatia was presented: Inocybe brijunica, I. istriaca, and Parasola papillatospora. An important biogeographical contribution was ten species of fungi found for the first time in Croatia: Coprinopsis alnivora, Entonaema cinnabarinum and 8 species of Parasola. Coprinopsis alnivora and P. malakandensis were also recorded for the first time in Europe. The integrative taxonomic analysis of the genus Entonaema led to the determination of their phylogenetic and taxonomic position in the family Hypoxylaceae and the establishment of an identification key of worldwide species within this genus

Selection of Lactobacillus and Enterococcus strain from dry sausages for their potential use as probiotics

Cilj ovog diplomskog rada bio je utvrditi probiotički potencijal vrsta Enterococcus durans (n = 4) i Lactobacillus sakei (n = 9) izoliranih iz tradicionalnih trajnih kobasica. Ispitan je sigurnosni status selektiranih sojeva, kao i njihova sposobnost autoagregacije i preživljavanja u simuliranim gastrointestinalnim uvjetima. Kod niti jednog od selektiranih izolata nije utvrđena rezistencija na korištene antibiotike i hemolitička aktivnost te nije detektirana prisutnost gena koji kodiraju za virulenciju ili produkciju biogenih amina. Svi selektirani sojevi pokazali su lošu sposobnost autoagregacije s prosječnom vrijednosti od 17,67 % nakon 5 h inkubacije. Najveći broj izolata preživio je u uvjetima usne šupljine, s prosječnom vrijednosti od 83,25 % dok je mogućnost preživljavanja značajno smanjena u simuliranim uvjetima želuca. Simulirani uvjeti dvanaesnika pokazali su se nepovoljnima za preživljenje većine izolata, s prosječnim vrijednostima preživljavanja od 0,75 % za sojeve L. sakei te 9,95 % za sojeve E. durans. Usprkos tomu, jedan soj E. durans (Ed3) pokazao je značajno više vrijednosti preživljavanja i potencijalni je kandidat za daljnja istraživanja vezana uz probiotičku primjenu.The aim of this master thesis was to evaluate probiotic potential of Enterococcus durans (n = 4) and Lactobacillus sakei (n = 9) isolated from Croatian traditional dry fermented sausages. Safty of the selected genotypes was investigated, as their ability go autoaggregate and survive under simulated gastrointestinal conditions. None of the selected genotypes showed resistance to tested antibiotics or hemolytic activity and the presence of the virulence determinants or genes encoding for production of biogenic amines were confirmed. All of the selected genotypes exhibited low ability to autoaggregate, with average value of 17.67 after 5 h of incubation. Regarding survival rates in simulated gastrointestinal conditions, all genotypes were best able to tolerate the conditions of oral cavity with average survival rate of 83.25 %. In the simulated environment of the stomach, the viability of bacteria significantly decreased, but the most detrimental effect had simulated conditions of small intestine with average values of 0.75 % for L. sakei strains and 9.95 % for E. durans. However, one E. durans genotype (Ed3) stands out with higher survival rates and can be considered for further analysis as a potential probiotic culture

Selection of Lactobacillus and Enterococcus strain from dry sausages for their potential use as probiotics

Cilj ovog diplomskog rada bio je utvrditi probiotički potencijal vrsta Enterococcus durans (n = 4) i Lactobacillus sakei (n = 9) izoliranih iz tradicionalnih trajnih kobasica. Ispitan je sigurnosni status selektiranih sojeva, kao i njihova sposobnost autoagregacije i preživljavanja u simuliranim gastrointestinalnim uvjetima. Kod niti jednog od selektiranih izolata nije utvrđena rezistencija na korištene antibiotike i hemolitička aktivnost te nije detektirana prisutnost gena koji kodiraju za virulenciju ili produkciju biogenih amina. Svi selektirani sojevi pokazali su lošu sposobnost autoagregacije s prosječnom vrijednosti od 17,67 % nakon 5 h inkubacije. Najveći broj izolata preživio je u uvjetima usne šupljine, s prosječnom vrijednosti od 83,25 % dok je mogućnost preživljavanja značajno smanjena u simuliranim uvjetima želuca. Simulirani uvjeti dvanaesnika pokazali su se nepovoljnima za preživljenje većine izolata, s prosječnim vrijednostima preživljavanja od 0,75 % za sojeve L. sakei te 9,95 % za sojeve E. durans. Usprkos tomu, jedan soj E. durans (Ed3) pokazao je značajno više vrijednosti preživljavanja i potencijalni je kandidat za daljnja istraživanja vezana uz probiotičku primjenu.The aim of this master thesis was to evaluate probiotic potential of Enterococcus durans (n = 4) and Lactobacillus sakei (n = 9) isolated from Croatian traditional dry fermented sausages. Safty of the selected genotypes was investigated, as their ability go autoaggregate and survive under simulated gastrointestinal conditions. None of the selected genotypes showed resistance to tested antibiotics or hemolytic activity and the presence of the virulence determinants or genes encoding for production of biogenic amines were confirmed. All of the selected genotypes exhibited low ability to autoaggregate, with average value of 17.67 after 5 h of incubation. Regarding survival rates in simulated gastrointestinal conditions, all genotypes were best able to tolerate the conditions of oral cavity with average survival rate of 83.25 %. In the simulated environment of the stomach, the viability of bacteria significantly decreased, but the most detrimental effect had simulated conditions of small intestine with average values of 0.75 % for L. sakei strains and 9.95 % for E. durans. However, one E. durans genotype (Ed3) stands out with higher survival rates and can be considered for further analysis as a potential probiotic culture