Sporobolomyces agrorum sp. Nov. and sporobolomyces sucorum sp. nov., two novel basidiomycetous yeast species isolated from grape and apple must in italy

During a survey of yeast populations associated with grape and apple musts used for wine and cider fermentation, respectively, six pink-coloured ballistoconidia-forming yeasts belonging to the order Sporidiobolales (Basidiomycota) were isolated. Phylogenetic analysis inferred using sequences of the internal transcribed spacer (ITS), the D1/D2 domain of the large subunit rRNA gene, the small subunit (SSU) rRNA gene and DNA-directed RNA polymerase II subunit (RPB2) indicated that the six isolates were separated in two novel species. One of the new species, Sporobolomyces agrorum sp. nov., isolated from grape must, had Sporobolomyces roseus and Sporobolomyces metaroseus as its closest relatives, but showed four/two and 16 nucleotide substitutions in the D1/D2 and ITS regions, respectively, to these two species. The other novel species, Sporobolomyces sucorum sp. nov., was found in apple must and was closely related to Sporobolomyces pararoseus and Sporobolomyces patagonicus, but showed two/three and five substitutions in those two regions for its closest relatives. We detected additional representatives of this species, most of them isolated from grapes whose sequences were already available on public databases. A sexual stage could not be observed for the novel species.publishersversionpublishe

A study on the yeast and mould biota associated with the production of Italian wines from post-harvest dried grapes

I vini Passiti ottenuti da uve che hanno subito un processo di appassimento sono vini rinomati e apprezzati dai consumatori principalmente per le complesse note sensoriali dovute al particolare processo di vinificazione. La fase di sovra-maturazione \ue8 fortemente influenzata dai microrganismi presenti sull\u2019acino e in particolare da Botrytis cinerea in forma nobile. La presenza di questa muffa favorisce la disidratazione dell\u2019acino e modifica la composizione del mosto che, infatti, presenta un elevato tenore zuccherino e contiene metaboliti secondari prodotti dalla muffa che possono inibire il processo fermentativo. Date queste condizioni stressanti per i lieviti, la fermentazione spontanea \ue8 spesso accompagnata da elevata produzione di acidit\ue0 volatile oltre che a blocchi o arresti fermentativi. Di conseguenza, per controllare meglio questa fase critica una strategia biotecnologica innovativa potrebbe essere l'utilizzo di colture starter di lieviti autoctoni ben adattati a queste condizioni. L\u2019obiettivo di questo progetto di ricerca \ue8 stato - quindi - quello di acquisire conoscenze sulla diversit\ue0 microbica associata alla produzione di alcuni vini italiani (quali Amarone, Vin Santo di Gambellara e Vino Santo Trentino) e di ottimizzare il processo di vinificazione attraverso l'uso di ceppi selezionati di B. cinerea e di lieviti autoctoni durante il processo di appassimento e la fermentazione. In questo contesto, \ue8 stata allestita un\u2019ampia collezione di muffe, tra cui B. cinerea, isolate da uve appassite, e una collezione di lieviti costituita da lieviti Saccharomyces e non-Saccharomyces isolati durante il processo di affinamento in caratelli e durante la fase di fermentazione di mosto Garganega utilizzato per la produzione del Vin Santo di Gambellara. Per quento riguarda l\u2019appassimento, a processo concluso, il vino ottenuto da queste uve \ue8 stato analizzato gas-cromatograficamente. I vini ottenuti hanno indicato differenze importanti tra le tesi inoculate e l'uva di controllo (non inoculata) per diversi composti volatili liberi. Come previsto, il livello pi\uf9 alto di 1-otten-3-olo (sapore di muffa) \ue8 stato trovato per i vini ottenuti da uve inoculato. Questi dati suggeriscono che l'inoculo di ceppi selezionati di B. cinerea su uva pu\uf2 influenzare le caratteristiche organolettiche del mosto/vino, consentendo cos\uec una diversificazione del prodotto finale. Per quanto riguarda le prove di fermentazione guidata, l'analisi dei vini ha rivelato che la fermentazione spontanea produce un elevato livello di acetato di etile rispetto alle tesi inoculate con i ceppi selezionati di S. cerevisiae. Pertanto, l'uso di ceppi autoctoni pu\uf2 limitare la produzione di acidit\ue0 volatile. Inoltre, l'analisi dei composti volatile ha evidenziato come tutti i vini ottenuti dai mosti fermentati con i ceppi selezionati presentano aromi associati a frutta bianca, alla rosa mandorla, fragola e caramello enfatizzando le propriet\ue0 aromatiche e il sapore del Vin Santo di Gambellara. L'analisi sensoriale ha ulteriormente confermato le differenze tra i diversi vini suggerendo quindi che l'uso di ceppi selezionati pu\uf2 migliorare il processo di fermentazione di vini passiti. In conclusione, questo progetto di ricerca ha evidenziato che l'uso di ceppi indigeni opportunamente selezionati (muffe e lieviti) \ue8 in grado di migliorare l\u2019intero processo di produzione dei vini passiti minimizzando i rischi di blocchi, arresti fermentativi e produzione di acidit\ue0 volatile, nonch\ue8 di influenzare positivamente la qualit\ue0 sensoriale del prodotto finito. Unitamente all\u2019uso di culture starter, l'uso di metodi alternativi validi in sostituzione all\u2019anidride solforosa (SO2) potrebbe migliorare l'impatto commerciale dei vini privi di conservanti chimici. In questo modo pu\uf2 essere soddisfatta la sempre pi\uf9 elevata domanda da parte dei consumatori di vini sicuri, salubri e organoletticamente distintivi.The wines obtained from post-harvest dried grapes are renowned wines, appreciated by consumers because of their complex sensorial notes due to the special vinification process, which involves the withering of grapes. The over-ripening phase is strongly influenced by the heterogeneous fungal biota present on the grape berry skin, and particularly by Botrytis cinerea. The development of the noble rot in berries affects the composition of the grape juice that presents high sugar content and inhibitory substances, as well. Therefore, grape juice fermentation proceeds hardly and is often accompanied by high production of volatile acidity. To better control the critical steps of Passito winemaking process, i.e. withering and fermentation, the use of suitable selected starter cultures could be applied as an innovative biotechnological strategy. Aims of this project were to increase knowledge about the diversity and evolution of indigenous eukaryotic microbiota associated with the production of some selected Italian wines (i.e. Amarone, Vin Santo di Gambellara, and Vino Santo Trentino) and to optimize the whole winemaking process through the use of selected indigenous strains of B. cinerea and yeasts. To these purposes, a large collection of moulds including B. cinerea was assembled; while a collection of yeasts (Saccharomyces and non-Saccharomyces) from wine aging in barrels and from the fermentation process of Garganega grape juice used for the production of Vin Santo di Gambellara was constituted. The isolates were identified using phenotypic and molecular methods, and then the collections were dereplicated by RAPD-PCR, PCR-RFLP, TEs, and microsatellite analysis. These techniques allowed the detection of a high genetic biodiversity among the B. cinerea and yeast isolates. The presence of distinctive strains could differentiate the withering of the studied grapes and the fermentation process, consequently selected strains of B. cinerea and yeasts were further characterized for several oenological properties (i.e. laccase activity and fungicide resistance for B. cinerea; resistance to stressful conditions and production of secondary metabolites for yeasts). In this way, strains of B. cinerea, S. cerevisiae and Zygosaccharomyces spp. were selected and tested in pilot-scale induction and fermentation trials, respectively. Considering the withering phase, at the end of the process the grapes were crushed and the grape juices were fermented. The gas-chromatographic analysis of the obtained wines indicated important differences between the inoculated and control grapes for several free volatile compounds. As expected, higher level of 1-octen-3-ol (mould flavor) was found for the wines obtained from the inoculated grape. These data suggest that the inoculum of selected B. cinerea strains on grapes influence the organoleptic characteristics of the must/wine, thus allowing a diversification of the final product. As regard the fermentation phase, the use of indigenous S. cerevisiae and non-Saccharomyces strains improved the fermentation process of Vin Santo di Gambellara. Indeed, failure to initiate fermentation and stuck fermentation were avoided, and a lower level of ethyl acetate was produced respect to the spontaneous fermentation. All selected S. cerevisiae yeasts produced flavors associated with white fruit, rose note, almond note and strawberry and caramel fragrance that emphasized the aromatic and flavor properties of this Passito wine. In conclusion, this research project suggests that the use of selected indigenous strains (moulds and yeasts) could improve the fermentation process of Passito wines minimizing risky conditions and improving the sensory quality of the final product. In other side, the use of valid alternative methods to SO2 addiction could improve the commercial impact of the wines free of chemical preservatives. In this way the modern consumer demand for wines that are safe and characterized by particular organoleptic flavour can be satisfied

Fungal Systematics and Evolution: FUSE 4

Eight new species presented are Calostoma areolatum collected in Wuyishan National Park (China), Crinipellis bidens from Hubei Province (China), Lactifluus sainii from Himalayan India, Inocybe elata from Yunnan (China), Inocybe himalayensis from Pakistan. Specimens previously identified as Massalongia carnosa represent a new species, namely M. patagonica restricted to southern South America. Saprolegnia maragheica is a new oomycete species of fresh water in Maraghe (Iran). Uncispora wuzhishanensis is a new aquatic hyphomycete species. A type specimen of Raddetes turkestanicus was studied and based on this the new combination Conocybe turkestanica, is proposed. Argyranthemum frutescens is a new host for Alternaria alternata and Syzygium cumini for Phyllosticta capitalensis in India. Crepidotus ehrendorferi is confirmed for Hungary and Pluteus leucoborealis for Central Europe, and for the phytogeographical region of Carpaticum. Pseudopithomyces palmicola is shown to occur on grapevine and it is validated by adding a unique identifier. Terfezia fanfani is reported first from Algeri

Epiphytic bacteria from withered grapes and their antagonistic effects on grape-rotting fungi.

The bacterial community in the surface of withered grapes, which are partially dehydrated in the post-harvest period to produce Italian passito wine, has been seldom investigated. Fifty epiphytic bacterial strains isolated from withered berries were identified and characterized. Genera such as Bacillus, Brevibacillus, Curtobacterium, Micrococcus, Pseudomonas and Staphylococcus have been identified by comparative sequence and phylogenetic analyses of 16S rRNA gene sequences. Bacillus was predominant and several taxa within this genus have been recognized. All isolates were characterized by PCR fingerprinting and assayed for osmotic tolerance, motility and antifungal activity. Several Bacillus strains displayed antagonistic effects on grape-rotting fungi such as Botrytis cinerea, Penicillium expansum and Aspergillus uvarum. The other strains were weakly or non-antagonistic on these fungi. Assay on antagonistic interactions among bacteria was also carried out. Bacillus strains, which exhibit swimming and swarming motility, have the potential to colonize the grape surface and to compete with their neighbours for space and resources. The occurrence of these isolates could reduce the contamination of fungal pathogens during grape withering. Epiphytic antagonistic bacteria could potentially be of interest for fungal biocontrol in the post-harvest processing of fruit and vegetables

Identification of Pestalotiopsis bicilita, Diplodia seriata and Diaporthe eres causing fruit rot in withered grapes in Italy

Pestalotiopsis biciliata, Diplodia seriata and Diaporthe eres were recovered from severely diseased bunches of withered grapes for Amarone wine production. To our knowledge this post-harvest disease has never been seen before in withered grapes. Fruit rot occurred with bunches containing decayed berries partially or totally covered by whit- ish mycelium. Morphological and phylogenetic analyses of fungal isolates from symptomatic fruits enabled species delimitation. Pathogenicity tests re- vealed that the three species are able to infect grape berries confirming that they are casual agents of fruit rot. This study provides preliminary insights on this post-harvest disease that might have important im- plications in grape quality

Description of a taxonomically undefined Sclerotiniaceae strain from withered rotten-grapes

A necrotrophic member of the Sclerotiniaceae family (herewith named strain C10) isolated from withered rotten-grapes, is described. Interestingly, the fungus has no defined taxonomic position since it has been impossible to attribute it to an existing genus. Phylogenetic analysis of partial sequences of glyceraldehyde 3-phosphate dehydrogenase (G3PDH), heat shock protein 60 (HSP60) and DNA-directed RNA polymerase II subunit (RPB2), revealed that strain C10 is distantly related to Amphobotrys and Botrytis. This evidence clearly distinguishes this new Sclerotiniaceae member from other taxa of the family. Moreover, its morphological characteristics did not match those of Amphobotrys and Botrytis. Infectivity assays demonstrated that strain C10 could be a potential postharvest pathogen of withered grapes. This study revealed the taxonomic importance of this strain suggesting the existence of a possible new genus, a theory that requires further investigation

Yeast-like fungi and yeasts in withered grape carposphere: Characterization of Aureobasidium pullulans population and species diversity

Yeast-like fungi and yeasts residing on carposphere of withered grapes for Italian passito wine production have been scarcely investigated. In the present study, isolates from single berries, both sound and damaged, of Nosiola, Corvina and Garganega varieties were analyzed at the end of the withering process. Great variation of cell concentration among single berries was observed. In sound berries, yeast-like fungi were significantly more frequent than yeasts. Species identification of isolates was carried out by BLAST comparative analysis on gene databases and phylogenetic approach. All yeast-like fungi isolates belonged to Aureobasidium pullulans. They displayed different culture and physiological characteristics and inhibitory capacity against phytopathogenic fungi. Moreover, PCR profile analysis revealed high genotypic similarity among these strains. A total of 35 species were recognized among yeast isolates. Ascomycetes prevailed over basidiomycetes. To the best of our knowledge, Naganishia onofrii and Rhodosporidiobolus odoratus were identified for the first time among yeasts isolated from grapes, must or wine. Hanseniaspora uvarum and Starmerella bacillaris were the most frequent species. Most species were found only in one grape variety (nine in Nosiola, 10 in Corvina and five in Garganega). The sanitary state of withered grapes could have an important impact on the structure of these epiphytic population

Indagini sulla micoflora presente nei fruttai per l\u2019appassimento delle uve e sugli effetti dei principali generi di fungi filamentosi sulla qualit\ue0 delle uve e del vino

In questo studio si descrivono le principali specie di funghi filamentosi isolati in 5 fruttai rappresentativi di ogni area produttiva (Valpolicella, Soave e Valle dei Laghi) durante l\u2019appassimento dell\u2019uva Corvina, Garganega e Nosiola per la produzione rispettivamente di Amarone, Recioto e Vino Santo. Le bacche, anche apparentemente sane, oltre alla botrite, ai penicilli e aspergilli, ospitano numerose specie che possono concorrere allo sviluppo dei marciumi dei grappoli. Per una gestione oculata del fruttaio \ue8 necessario non sottovalutare la loro presenza, che in favorevoli condizioni ambientali, posso causare contaminazioni signifi cative a detrimento dell\u2019uva.In this study, the main species of filamentous fungi were described. These fungi were isolated from withered grapes (Corvina, Garganega and Nosiola) used to produce Amarone, Recioto e Vino Santo wines located in five representative fruit-drying room in Valpolicella, Soave e Valle dei Laghi areas. The berries, healthy and injured, can host numerous fungi species such as Penicillium and Aspergillus, as well as Botrytis, that are correlated to the rots onto grape berries. To improve the grape management during the withering process it is better do not disregard their occurrence, that in specific environmental condition can cause important department e thus detriment of grape

Isolation of Neofusicoccum parvum from withered grapes: Strain characterization, pathogenicity and its detrimental effects on passito wine arom

Aims: There is scarce information on the occurrence of several fungi that infect withered grapes to produce passito wine. Isolation and characterization of Neofusicoccum parvum strains and evaluation of their effects on withered grape and wine were carried out.Methods and Results: Nine isolates were phenotypically characterized by colony morphology and genetically discriminated by molecular methods. Two representative strains were identified as N.parvum according to the phylogenetic analysis of internal transcribed spacer (ITS), and a part of translation elongation factor 1-alfa (TEF) and -tubulin DNA sequences. The pathogenicity of both strains on grape berries varied according to the inoculation and incubation conditions. Under withering conditions, infected berries showed browning and shrivelling and some berries showed pycnidial development on the surface. The infection affected laccase, esterase, -glucosidase and tannase on grape juice as well as the content of several aroma molecules on resulting wines. Strain-specific effects on wine composition were also observed.Conclusions: Neofusicoccum parvum occurred in withered grapes and was able to infect grapes under withering condition changing the aroma wine.Significance and Impact of the Study: This study reports for the first time the N.parvum isolation in fruit-drying rooms and indicates its important role on postharvest grape infection

Yeast species diversity in apple juice for cider production evidenced by culture-based method

Identification of yeasts isolated from apple juices of two cider houses (one located in a plain area and one in an alpine area) was carried out by culture-based method. Wallerstein Laboratory Nutrient Agar was used as medium for isolation and preliminary yeasts identification. A total of 20 species of yeasts belonging to ten different genera were identified using both BLAST algorithm for pairwise sequence comparison and phylogenetic approaches. A wide variety of non-Saccharomyces species was found. Interestingly, Candida railenensis, Candida cylindracea, Hanseniaspora meyeri, Hanseniaspora pseudoguilliermondii, and Metschnikowia sinensis were recovered for the first time in the yeast community of an apple environment. Phylogenetic analysis revealed a better resolution in identifying Metschnikowia and Moesziomyces isolates than comparative analysis using the GenBank or YeastIP gene databases. This study provides important data on yeast microbiota of apple juice and evidenced differences between two geographical cider production areas in terms of species composition