Effect of Debaryomyces hansenii and the antifungal PgAFP protein on Alternaria spp. growth, toxin production, and RHO1 gene expression in a tomato-based medium.

Tomato fruit is susceptible to Alternaria spp. spoilage, which poses a health risk due to their mycotoxin production. Biopreservation relies on the use of whole microorganisms or their metabolites to manage spoilage microorganisms including filamentous fungi. However, the use of treatments at fungistatic level might activate intracellular pathways, which can cause an increment in mycotoxin accumulation. The objective of this work was to evaluate the effect of two strains of Debaryomyces hansenii and the antifungal protein PgAFP at 10 and 40 µg/mL. Both growth and production of two of the most common mycotoxins (tenuazonic acid and alternariol monomethyl ether) by Alternaria tenuissima sp.-grp. and Alternaria arborescens sp.-grp. on a tomato-based matrix, were analysed at 12 °C. Additionally, the impact of these biocontrol agents on the stress-related RHO1 gene expression was assessed. All treatments reduced mycotoxin accumulation (from 27 to 92% of inhibition). Their mode of action against Alternaria spp. in tomato seems unrelated to damages to fungal cell wall integrity at the genomic level. Therefore, the two D. hansenii strains (CECT 10352 and CECT 10353) and the antifungal protein PgAFP at 10 µg/mL are suggested as biocontrol strategies in tomato fruit at postharvest stage.

Metschnikowia pulcherrima represses aerobic respiration in Saccharomyces cerevisiae suggesting a direct response to co-cultivation.

The use of non-Saccharomyces species as starter cultures together with Saccharomyces cerevisiae is becoming a common practice in the oenological industry to produce wines that respond to new market demands. In this context, microbial interactions with these non-Saccharomyces species must be considered for a rational design of yeast starter combinations. Previously, transcriptional responses of S. cerevisiae to short-term co-cultivation with Torulaspora delbrueckii, Candida sake, or Hanseniaspora uvarum was compared. An activation of sugar consumption and glycolysis, membrane and cell wall biogenesis, and nitrogen utilization was observed, suggesting a metabolic boost of S. cerevisiae in response to competing yeasts. In the present study, the transcription profile of S. cerevisiae was analyzed after 3 h of cell contact with Metschnikowia pulcherrima. Results show an over-expression of the gluco-fermentative pathway much stronger than with the other species. Moreover, a great repression of the respiration pathway has been found in response to Metschnikowia. Our hypothesis is that there is a direct interaction stress response (DISR) between S. cerevisiae and the other yeast species that, under excess sugar conditions, induces transcription of the hexose transporters, triggering glucose flow to fermentation and inhibiting respiration, leading to an increase in both, metabolic flow and population dynamics.

On the use of n-octyl gallate and salicylhydroxamic acid to study the alternative oxidase role.

The alternative oxidase (AOX) catalyzes the transfer of electrons from ubiquinol to oxygen without the translocation of protons across the inner mitochondrial membrane. This enzyme has been proposed to participate in the regulation of cell growth, sporulation, yeast-mycelium transition, resistance to reactive oxygen species, infection, and production of secondary metabolites. Two approaches have been used to evaluate AOX function: incubation of cells for long periods of time with AOX inhibitors or deletion of AOX gene. However, AOX inhibitors might have different targets. To test non-specific effects of n-octyl gallate (nOg) and salicylhydroxamic acid (SHAM) on fungal physiology we measured the growth and respiratory capacity of two fungal strains lacking (Ustilago maydis-Δaox and Saccharomyces cerevisiae) and three species containing the AOX gene (U. maydis WT, Debaryomyces hansenii, and Aspergillus nidulans). For U. maydis, a strong inhibition of growth and respiratory capacity by SHAM was observed, regardless of the presence of AOX. Similarly, A. nidulans mycelial growth was inhibited by low concentrations of nOg independently of AOX expression. In contrast, these inhibitors had no effect or had a minor effect on S. cerevisiae and D. hansenii growth. These results show that nOg and SHAM have AOX independent effects which vary in different microorganisms, indicating that studies based on long-term incubation of cells with these inhibitors should be considered as inconclusive.

Genome-wide identification, characterization and expression analysis of lineage-specific genes within Hanseniaspora yeasts.

Lineage-specific genes (LSGs) are defined as genes with sequences that are not significantly similar to those in any other lineage. LSGs have been proposed, and sometimes shown, to have significant effects in the evolution of biological function. In this study, two sets of Hanseniaspora spp. LSGs were identified by comparing the sequences of the Kloeckera apiculata genome and of 80 other yeast genomes. This study identified 344 Hanseniaspora-specific genes (HSGs) and 109 genes ('orphan genes') specific to K. apiculata. Three thousand three hundred thirty-one K. apiculata genes that showed significant similarity to at least one sequence outside the Hanseniaspora were classified into evolutionarily conserved genes. We analyzed their sequence features, functional categories, gene origin, gene structure and gene expression. We also investigated the predicted cellular roles and Gene Ontology categories of the LSGs using functional inference. The patterns of the functions of LSGs do not deviate significantly from genome-wide average. The results showed that a few LSGs were formed by gene duplication, followed by rapid sequence divergence. Many of the HSGs and orphan genes exhibited altered expression in response to abiotic stress. Studying these LSGs might be helpful for understanding the molecular mechanism of yeast adaption.

Melatonin and glycolytic protein interactions are related to yeast fermentative capacity.

Melatonin is an indole amine that interacts with some proteins in mammals, such as calreticulin, calmodulin or sirtuins. In yeast, melatonin is synthetized and interacts with glycolytic proteins during alcoholic fermentation in Saccharomyces cerevisiae. Due to its importance as an antioxidant molecule in both Saccharomyces and non-Saccharomyces yeasts, the aim of this study was to determine the intracellular and extracellular synthesis profiles of melatonin in four non-Saccharomyces strains (Torulaspora delbrueckii, Hanseniaspora uvarum, Starmeralla bacillaris and Metschnikowia pulcherrima) and to confirm whether glycolytic enzymes can also interact with this molecule in non-conventional yeast cells. Melatonin from fermentation samples was analyzed by liquid chromatography mass spectrometry, and proteins bound to melatonin were immunopurified by melatonin-IgG-Dynabeads. Melatonin was produced in a similar pattern in all non-Saccharomyces yeast, with M. pulcherrima and S. bacillaris being the highest producers. However, melatonin only bound to proteins in two non-conventional yeasts, S. bacillaris and T. delbrueckii, which specifically had higher fermentative capacities. Sequence analysis showed that most proteins shared high levels of homology with glycolytic enzymes, but an RNA-binding protein, the elongation alpha factor, which is related to mitochondria, was also identified. This study reports for the first time the interaction of melatonin with proteins inside non-Saccharomyces yeast cells. These results reinforce the possible role of melatonin as a signal molecule, likely related to fermentation metabolism and provide a new perspective for understanding its role in yeast.

Overlapping responses between salt and oxidative stress in Debaryomyces hansenii.

Debaryomyces hansenii is a halotolerant yeast of importance in basic and applied research. Previous reports hinted about possible links between saline and oxidative stress responses in this yeast. The aim of this work was to study that hypothesis at different molecular levels, investigating after oxidative and saline stress: (i) transcription of seven genes related to oxidative and/or saline responses, (ii) activity of two main anti-oxidative enzymes, (iii) existence of common metabolic intermediates, and (iv) generation of damages to biomolecules as lipids and proteins. Our results showed how expression of genes related to oxidative stress was induced by exposure to NaCl and KCl, and, vice versa, transcription of some genes related to osmotic/salt stress responses was regulated by H2O2. Moreover, and contrary to S. cerevisiae, in D. hansenii HOG1 and MSN2 genes were modulated by stress at their transcriptional level. At the enzymatic level, saline stress also induced antioxidative enzymatic defenses as catalase and glutathione reductase. Furthermore, we demonstrated that both stresses are connected by the generation of intracellular ROS, and that hydrogen peroxide can affect the accumulation of in-cell sodium. On the other hand, no significant alterations in lipid oxidation or total glutathione content were observed upon exposure to both stresses tested. The results described in this work could help to understand the responses to both stressors, and to improve the biotechnological potential of D. hansenni.

Characterization of tolerance and multi-enzyme activities in non-Saccharomyces yeasts isolated from Vidal blanc icewine fermentation.

This work aim to study the tolerance and aroma-related enzymes activities of the non-Saccharomyces yeasts in Vidal blanc icewine from the Huanren region of China. The strains were identified by sequencing internal transcribed spacer (ITS) region and the 26S rDNA D1/D2 domain genes and all representative non-Saccharomyces yeasts were belonged to genera Metschnikowia, Hanseniaspora, Torulaspora, Candida, and Debaryomyces. A total of 28 strains were carried out for tolerance experiments and results suggested that most of them could tolerate 500 g/L glucose, 4% ethanol, 20 g/L tartaric acid, and 350 mg/L SO2 . Finally, a total of 17 strains with better tolerance were carried out for the ß-glucosidase, ß-xylosidase, and pectinase activities experiments. The results showed that Candida railenensis HC08 and the strains of Hanseniaspora genus have satisfactory multi-enzyme activities, which can be used to design mixed fermentation to produce characteristic icewine. PRACTICAL APPLICATIONS: Non-Saccharomyces yeasts produces a series of hydrolytic enzymes that are thought to have a significant contribution to the aroma complexity of wines, however, are poorly explored in icewine. In this work, most of the non-Saccharomyces yeasts screened from Chinese icewine can adapt well to the high-sugar and high-acid environment of icewine, and can secrete hydrolase. The application of these strains in mixed fermentation could provide a prospect for the production of characteristic icewine.

Debaryomyces hansenii Metabolism of Sulfur Amino Acids As Precursors of Volatile Sulfur Compounds of Interest in Meat Products.

The ability of Debaryomyces hansenii to produce volatile sulfur compounds from sulfur amino acids and the metabolic pathway involved have been studied in seven strains from different food origins. Our results proved that l-methionine is the main precursor for sulfur compound generation. Crucial differences in the sulfur compound profile and amino acid consumption among D. hansenii strains isolated from different food sources were observed. Strains isolated from dry pork sausages displayed the most complex sulfur compound profiles. Sulfur compound production, such as that of methional, could result from chemical reactions or yeast metabolism, while according to this study, thioester methyl thioacetate appeared to be generated by yeast metabolism. No relationship between sulfur compounds production by D. hansenii strains and the expression of genes involved in sulfur amino acid metabolism was found, except for the ATF2 gene in the L1 strain for production of methyl thioacetate. Our results suggest a complex scenario during sulfur compound production by D. hansenii.

Quantitative proteomic profiling of ochratoxin A repression in Penicillium nordicum by protective cultures.

Dry-cured meat products are usually contaminated with moulds during ripening. Although fungal development contributes to the desired sensory characteristics, some moulds, such as Penicillium nordicum are able to produce ochratoxin A (OTA) on meat products. Therefore, strategies to prevent OTA contamination in ripened meat products are required. Microorganisms isolated from these meat products can be adequate as biocontrol agents, given that no negative sensory impact is expected. The PgAFP antifungal protein-producer Penicillium chrysogenum (Pc) and Debaryomyces hansenii (Dh) have been shown to successfully inhibit toxigenic moulds. However, scarce information about the mechanism of action of these biocontrol agents on toxigenic mould inhibition is available. Comparative proteomic analysis is a powerful tool to investigate the physiological response of microorganisms to stimuli. Proteomic analysis was carried out on P. nordicum co-cultured with Pc, Dh, PgAFP, and their combinations on a dry-cured ham-based medium. Additionally, OTA production by P. nordicum in the different cultures was measured. The individual inoculation of Pc or Dh repressed OTA production by P. nordicum by 5 and 3.15 fold, respectively. A total of 2844 unique P. nordicum proteins were identified by proteomic analysis. The impact of the biocontrol agents on the proteome of P. nordicum was higher for Pc-containing cultures, followed by Dh-containing treatments. PgAFP alone had minimal impact on the proteome of P. nordicum. Proteomic analyses indicated Pc repressed P. nordicum OTA production through nutrient competition, potentially reducing glucose availability. Data also suggest that Dh and Pc inhibited P. nordicum through cell wall integrity impairment. Both Pc and Dh seem to hamper P. nordicum secondary metabolism (SM) as indicated by lower levels of MAP kinases and SM-associated proteins found in the co-inoculated P. nordicum. This work paves the way to use antifungal agents in the most efficient way to prevent OTA formation in meat products.

Biocontrol of Penicillium griseofulvum to reduce cyclopiazonic acid contamination in dry-fermented sausages.

Dry-fermented sausages are very appreciated by consumers. The environmental conditions during its ripening favor colonization of their surface by toxigenic molds. These molds contribute to the development of sensory characteristics; however, some of them could produce mycotoxins such as cyclopiazonic acid (CPA). CPA is mainly produced by Penicillium commune and Penicillium griseofulvum which have been found in dry-cured meat products. Thus, strategies to prevent the CPA contamination in dry-fermented sausages are needed. The objective of this work was to evaluate the ability of P. griseofulvum to produce CPA in dry-fermented sausage during its ripening as well as to test different strategies to prevent CPA production. The ability of PgAFP antifungal protein-producing Penicillium chrysogenum, Debaryomyces hansenii and Pediococcus acidilactici for inhibiting CPA production by P. griseofulvum was tested on dry-fermented sausage-based medium. Only P. chrysogenum inhibited the CPA production, so this mold was co-inoculated with P. griseofulvum on sausages whose ripening was performed at low temperature. CPA reached around 800 ng/g in the control batch, being reduced to 20 ng/g by the presence of P. chrysogenum. This work demonstrates the risk posed by CPA on dry-fermented sausages, and provides a successful strategy to prevent this hazard.

Proteomics profile of Hanseniaspora uvarum enhanced with trehalose involved in the biocontrol efficacy of grape berry.

This present study tested the extent to which 2% w/v trehalose enhanced the proteins expression profile of Hanseniaspora uvarum Y3. Furthermore, it explored the relative gene expression of stilbene synthase (StSy), one of the vital defense-related genes found in the skin of grapes. The proteomics profile revealed that 29 proteins were differentially expressed out of which 26 were significantly up-regulated and 3 were download-regulated. The pathogenesis related (PR) and other protein spots were visible at 97.4 kDa and 14.4 kDa. Peroxiredoxin TSA1 and superoxide dismutase were the main proteins involved in defense response and both proteins were significantly up-regulated. The carbohydrate and energy metabolism proteins were also significantly up-regulated. The results revealed that the treatments were associated with substantial increase in peroxidase activity compared to the control. StSy relative gene expression level was observed to increase by 2.5-fold in grapes treated with the pre-enhanced H. uvarum compared to the control.

Inventory of ABC proteins and their putative role in salt and drug tolerance in Debaryomyces hansenii.

ATP-binding cassette (ABC) is one of the largest superfamily of proteins, which are ubiquitously present, performing variety of cellular functions. These proteins as drug transporters have been enticing substantial consideration because of their clinical importance. The present study focuses on genome wide identification of ABC proteins of an important halotolerant yeast Debaryomyces hansenii and explores their role in salt and drug tolerance. Our bioinformatics analysis identified a total of 30 putative ABC protein-coding genes whose expression at transcript level was confirmed by qRT-PCR. Our comparative phylogenetic analysis of nucleotide binding domains of D. hansenii and topology prediction categorized these proteins into six subfamilies; ABCB/MDR, ABCC/MRP, ABCD/ALDP, ABCF/YEF3, ABCE/RLI, and ABCG/PDR based on the nomenclature adopted by the Human Genome Organization (HUGO). Further, our transmembrane domain (TMD) predictions suggest that out of 30 ABC proteins, only 22 proteins possess either two or one TMD and hence are considered as membrane localized ABC proteins. Notably, our transcriptional dynamics of ABC proteins encoding genes following D. hansenii cells treatment with different salts and drugs concentrations illustrated variable transcriptional response of some of the genes, pointing to their role in salt and drug tolerance. This study first time provides a comprehensive inventory of the ABC proteins of a haploid D. hansenii which will be helpful for exploring their functional relevance.

Non-canonical Activities of Hog1 Control Sensitivity of Candida albicans to Killer Toxins From Debaryomyces hansenii.

Certain yeasts secrete peptides known as killer toxins or mycocins with a deleterious effect on sensitive yeasts or filamentous fungi, a common phenomenon in environmental species. In a recent work, different Debaryomyces hansenii (Dh) strains isolated from a wide variety of cheeses were identified as producing killer toxins active against Candida albicans and Candida tropicalis. We have analyzed the killer activity of these toxins in C. albicans mutants defective in MAPK signaling pathways and found that the lack of the MAPK Hog1 (but not Cek1 or Mkc1) renders cells hypersensitive to Dh mycocins while mutants lacking other upstream elements of the pathway behave as the wild type strain. Point mutations in the phosphorylation site (T174A-176F) or in the kinase domain (K52R) of HOG1 gene showed that both activities were relevant for the survival of C. albicans to Dh killer toxins. Moreover, Hog1 phosphorylation was also required to sense and adapt to osmotic and oxidative stress while the kinase activity was somehow dispensable. Although the addition of supernatant from the killer toxin- producing D. hansenii 242 strain (Dh-242) induced a slight intracellular increase in Reactive Oxygen Species (ROS), overexpression of cytosolic catalase did not protect C. albicans against this mycocin. This supernatant induced an increase in intracellular glycerol concentration suggesting that this toxin triggers an osmotic stress. We also provide evidence of a correlation between sensitivity to Dh-242 killer toxin and resistance to Congo red, suggesting cell wall specific alterations in sensitive strains.

Hanseniaspora thailandica BC9 ß-Glucosidase for the Production of ß-D-Hexyl Glucoside.

For biotechnological production of high-valued ß-D-hexyl glucoside, the catalytic properties of Hanseniaspora thailandica BC9 ß-glucosidase purified from the periplasmic fraction were studied, and the transglycosylation activity for the production of ß-D-hexyl glucoside was optimized. The constitutive BC9 ß-glucosidase exhibited maximum specific activity at pH 6.0 and 40ºC, and the activity of BC9 ß-glucosidase was not significantly inhibited by various metal ions. BC9 ß-glucosidase did not show a significant activity of cellobiose hydrolysis, but the activity was rather enhanced in the presence of sucrose and medium-chain alcohols. BC9 ß-glucosidase exhibited enhanced production of ß-D-hexyl glucoside in the presence of DMSO, and 62% of ß-D-hexyl glucoside conversion was recorded in 4 h in the presence of 5% 1-hexanol and 15% DMSO.

Glycolytic Functions Are Conserved in the Genome of the Wine Yeast Hanseniaspora uvarum, and Pyruvate Kinase Limits Its Capacity for Alcoholic Fermentation.

Hanseniaspora uvarum (anamorph Kloeckera apiculata) is a predominant yeast on wine grapes and other fruits and has a strong influence on wine quality, even when Saccharomyces cerevisiae starter cultures are employed. In this work, we sequenced and annotated approximately 93% of the H. uvarum genome. Southern and synteny analyses were employed to construct a map of the seven chromosomes present in a type strain. Comparative determinations of specific enzyme activities within the fermentative pathway in H. uvarum and S. cerevisiae indicated that the reduced capacity of the former yeast for ethanol production is caused primarily by an ∼10-fold-lower activity of the key glycolytic enzyme pyruvate kinase. The heterologous expression of the encoding gene, H. uvarumPYK1 (HuPYK1), and two genes encoding the phosphofructokinase subunits, HuPFK1 and HuPFK2, in the respective deletion mutants of S. cerevisiae confirmed their functional homology.IMPORTANCEHanseniaspora uvarum is a predominant yeast species on grapes and other fruits. It contributes significantly to the production of desired as well as unfavorable aroma compounds and thus determines the quality of the final product, especially wine. Despite this obvious importance, knowledge on its genetics is scarce. As a basis for targeted metabolic modifications, here we provide the results of a genomic sequencing approach, including the annotation of 3,010 protein-encoding genes, e.g., those encoding the entire sugar fermentation pathway, key components of stress response signaling pathways, and enzymes catalyzing the production of aroma compounds. Comparative analyses suggest that the low fermentative capacity of H. uvarum compared to that of Saccharomyces cerevisiae can be attributed to low pyruvate kinase activity. The data reported here are expected to aid in establishing H. uvarum as a non-Saccharomyces yeast in starter cultures for wine and cider fermentations.

Molecular characterization of Aspergillus fumigatus TcsC, a characteristic type III hybrid histidine kinase of filamentous fungi harboring six HAMP domains.

The type III hybrid histidine kinase (HHK) TcsC enables the pathogenic mold Aspergillus fumigatus to thrive under hyperosmotic conditions. It is, moreover, of particular interest, since it is the target of certain antifungal agents, such as fludioxonil. This study was aimed at a functional characterization of the domains that constitute the sensing and the kinase module of TcsC. The sensing module consists of six HAMP domains, an architecture that is commonly found in type III HHKs of filamentous fungi. To dissect the functional role of the individual domains, we have analyzed a set of truncated derivatives of TcsC with respect to their impact on fungal growth and their ability to respond to hyperosmotic stress and fludioxonil. Our data demonstrate that the TcsC kinase module per se is constitutively active and under the control of the sensing module. We furthermore found that the sixth HAMP domain alone is sufficient to arrest the kinase module in an inactive state. This effect can be partially lifted by the presence of the fifth HAMP domain. Constructs harboring more than these two HAMP domains are per se inactive and all six HAMP domains are required to enable a response to fludioxonil or hyperosmotic stress. When expressed in an A. fumigatus wild type strain, the construct harboring only the sixth HAMP domain exerts a strong dominant negative effect on the native TcsC. This effect is successively reduced in other constructs harboring increasing numbers of HAMP domains. To our knowledge, this is the first molecular characterization of a type III HHK containing six HAMP domains. Our data strongly suggest that TcsC is a positive regulator of its MAPK SakA and thereby differs fundamentally from the prototypic yeast type III HHK DhNik1 of Debaryomyces hansenii, which harbors only five HAMP domains and acts as a negative regulator of its MAPK.

The serine/threonine phosphatase DhSIT4 modulates cell cycle, salt tolerance and cell wall integrity in halo tolerant yeast Debaryomyces hansenii.

The highly conserved family of Phosphoprotein phosphatases (PPP) regulates several major physiological processes in yeast. However, very little is known about the PPP orthologs from the yeast species inhabiting extreme environmental niches. In the present study we have identified DhSIT4, a member of PPP6 class of serine threonine phosphatases from the halotolerant yeast Debaryomyces hansenii. Deletion of DhSIT4 in D. hansenii was not lethal but the mutant exhibited reduced growth due to its effect on the cell cycle. The knock out mutant Dhsit4Δ showed sensitivity towards Li+, Na+ and cell wall damaging agents. The expression of DhSit4p rescued salt, caffeine and calcofluor white sensitivity of Dhmpk1Δ strain and thereby indicating a genetic interaction of this phosphatase with the cell wall integrity pathway in this species. Our study also demonstrated the antagonistic roles of DhSit4p and DhPpz1p in maintaining the cell cycle and ion homeostasis in D. hansenii.

The yeast Scheffersomyces amazonensis is an efficient xylitol producer.

This study assessed the efficiency of Scheffersomyces amazonensis UFMG-CM-Y493T, cultured in xylose-supplemented medium (YPX) and rice hull hydrolysate (RHH), to convert xylose to xylitol under moderate and severe oxygen limitation. The highest xylitol yields of 0.75 and 1.04 g g-1 in YPX and RHH, respectively, were obtained under severe oxygen limitation. However, volumetric productivity in RHH was ninefold decrease than that in YPX medium. The xylose reductase (XR) and xylitol dehydrogenase (XDH) activities in the YPX cultures were strictly dependent on NADPH and NAD+ respectively, and were approximately 10% higher under severe oxygen limitation than under moderate oxygen limitation. This higher xylitol production observed under severe oxygen limitation can be attributed to the higher XR activity and shortage of the NAD+ needed by XDH. These results suggest that Sc. amazonensis UFMG-CM-Y493T is one of the greatest xylitol producers described to date and reveal its potential use in the biotechnological production of xylitol.

Detection of Multiple Budding Yeast Cells and a Partial Sequence of 43-kDa Glycoprotein Coding Gene of Paracoccidioides brasiliensis from a Case of Lacaziosis in a Female Pacific White-Sided Dolphin (Lagenorhynchus obliquidens).

Lacaziosis, formerly called as lobomycosis, is a zoonotic mycosis, caused by Lacazia loboi, found in humans and dolphins, and is endemic in the countries on the Atlantic Ocean, Indian Ocean and Pacific Ocean of Japanese coast. Susceptible Cetacean species include the bottlenose dolphin (Tursiops truncatus), the Indian Ocean bottlenose dolphin (T. aduncus), and the estuarine dolphin (Sotalia guianensis); however, no cases have been recorded in other Cetacean species. We diagnosed a case of Lacaziosis in a Pacific white-sided dolphin (Lagenorhynchus obliquidens) nursing in an aquarium in Japan. The dolphin was a female estimated to be more than 14 years old at the end of June 2015 and was captured in a coast of Japan Sea in 2001. Multiple, lobose, and solid granulomatous lesions with or without ulcers appeared on her jaw, back, flipper and fluke skin, in July 2014. The granulomatous skin lesions from the present case were similar to those of our previous cases. Multiple budding and chains of round yeast cells were detected in the biopsied samples. The partial sequence of 43-kDa glycoprotein coding gene confirmed by a nested PCR and sequencing, which revealed a different genotype from both Amazonian and Japanese lacaziosis in bottlenose dolphins, and was 99 % identical to those derived from Paracoccidioides brasiliensis; a sister fungal species to L. loboi. This is the first case of lacaziosis in Pacific white-sided dolphin.