Screening and identification of yeast strains from fruits and vegetables: Potential for biological control of postharvest chilli anthracnose (Colletotrichum capsici)

Yeasts antagonistic to Colletotrichum capsici were isolated from Thai fruits and vegetables. Four antagonists (R13, R6, ER1, and L2) were found that inhibited C. capsici growth with biocontrol efficacies of 93.3%, 83.1%, 76.6%, and 66.4%, respectively. Identification by 26S rDNA, and ITS region sequence together with physiological and morphological characteristics, showed them to be Pichia guilliermondii, Candida musae, Issatchenkia orientalis, and Candida quercitrusa, in order of their efficacy. P. guilliermondii strain R13 showed efficacy in reducing disease incidence on C. capsici infected chilli fruits to as low as 6.5%. Lower disease incidence was observed at lower storage temperature. The application of P. guilliermondii is more effective for preserving chilli fruits than conventional preservation with chlorinated water.     

Screening and identification of epiphytic yeasts with potential for biological control of green mold of citrus fruits

Epiphytic yeasts isolated from the surface of citrus fruits, harvested in several orchards in the Souss-Massa-Draa Valley, Agadir, Morocco, were in vivo screened for antagonistic activity against Penicillium digitatum, the causal agent of green mold of citrus. From a total of 245 yeast strains assessed for their biocontrol activity against P. digitatum, fifteen reduced the incidence of disease to less than 50%. The effectiveness of the best selected yeast strains showed that Pichia anomala (YT73), Debaryomyces hansenii (YT22) and Hanseniaspora guilliermondii (YT13) were the most effective, with a reduction of green mold incidence from 65 to ~80%, compared to the control. The identification of the fifteen selected yeast strains was carried out through an integrated approach including phenotypic and genotypic (sequencing of D1/D2 domain of 26S rDNA encoding gene) methods. These 15 selected were identified as: H. guilliermondii, D. hansenii, H. uvarum and P. anomala. The study of the dynamics of two of the best strains, H. guilliermondii and D. hansenii, showed that these strains can grow rapidly, by approximately 2 log units, in citrus fruit wounds. Such rapid growth in wounds indicates that these antagonist yeasts are excellent colonizers of citrus wounds and can thrive on citrus fruits as a substrate.     

Cloning and characterization of the inulinase gene from a marine yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis> and its expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The extracellular inulinase structural gene was isolated from the genomic DNA of the marine yeast Pichia guilliermondii strain 1 by PCR. The gene had an open reading frame of 1,542 bp long encoding an inulinase. The coding region of the gene was not interrupted by any intron. It encoded 514 amino acid residues of a protein with a putative signal peptide of 18 amino acids and the calculated molecular mass of 58.04 kDa. The protein sequence deduced from the inulinase structural gene contained the inulinase consensus sequences (WMNXPNGL) and (RDPKVF). It also had ten conserved putative N-glycosylation sites. The inulinase from P. guilliermondii strain 1 was found to be closely related to that from Kluyveromyces marxianus. The inulinase gene without the signal sequence was subcloned into pPICZαA expression vector and expressed in Pichia pastoris X-33. The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 60 kDa was found. Enzyme activity assay verified the recombinant protein as an inulinase. A maximum activity of 58.7 ± 0.12 U/ml was obtained from the culture supernatant of P. pastoris X-33 harboring the inulinase gene. A large amount of monosaccharides, disaccharides and oligosaccharides were detected after the hydrolysis of inulin with the crude recombinant inulinase.     

Biodegradation of VOCs from printing press air by an on-site pilot plant bioscrubber and laboratory scale continuous yeast cultures

The volatile organic compound composition (VOCs) of printing press air wasfound to contain mostly ethanol, but also ethyl acetate, 1-propanol, 2-propanol,1-methoxy-2-propanol and 3-ethoxy-1-propanol. A pilot plant bioscrubber inoculatedwith a mixed microbial population was constructed on-site. The bioscrubber wasable to treat the polluted gas efficiently. It, however, suffered from strong wall growthand blockages in the column. The efficiencies of the pilot plant and a bioreactor iscompared. The yeasts Candida guilliermondii and Saccharomyces cerevisiae known to tolerate ethanol were selected instead of mixed population to avoid the wall growth and blockages in the bioreactor. The removal of the VOCs both individually and as a complex mixture was tested in a microcultivation system and in continuous chemostat cultures with and without cell recycling. The Candida yeast could use all the compounds as a carbon source while growth of S. cerevisiae was markedly slower on the methoxylated and ethoxylated propanols. Best total removal of the VOCs was 99% and achieved by C. guilliermondii. The only compound that was not totally removable in the chemostat experiment with C. guilliermondii was 1-methoxy-2-propanol. In laboratory scale the total and volumetric removal of VOCs by C. guilliermondii was more efficient compared to the pilot plant encouraging to scale up and applying the yeast bioreactor to real field conditions.     

Effect of the rib83Mutation on Riboflavin Synthesis and Iron Acquisition in the Yeast Pichia guilliermondii

Abstract–Monogenicrib83mutation blocked riboflavin oversynthesis in the yeast Pichia guilliermondiiand lowered iron acquisition by cells, their ferric reductase activity, and the growth rate in iron-deficient media. Mutants with the combined mutations of rib83with rib80and rib81(the last two mutations impair the negative control of riboflavin synthesis and thus cause its oversynthesis) were unable to depress the enzymes of flavinogenesis (GTP cyclohydrolase and riboflavin synthase) or overproduce riboflavin in both iron-deficient and iron-sufficient media. This suggests that rib83mutation is epistatic with respect to rib80and rib81mutations. The RIB83gene may positively control both riboflavin synthesis and iron acquisition in the yeast P. guilliermondii.     

Cloning and expression of Candida guilliermondii xylose reductase gene (xyl1) in Pichia pastoris

A xylose reductase gene (xyl1) of Candida guilliermondii ATCC 20118 was cloned and characterized. The open reading frame of xyl1 contained 954 nucleotides encoding a protein of 317 amino acids with a predicted molecular mass of 36 kDa. The derived amino acid sequence of C. guilliermondii xylose reductase was 70.4% homologous to that of Pichia stipitis. The gene was placed under the control of an alcohol oxidase promoter (AOX1) and integrated into the genome of a methylotrophic yeast, Pichia pastoris. Methanol induced the expression of the 36-kDa xylose reductase in both intracellular and secreted expression systems. The expressed enzyme preferentially utilized NADPH as a cofactor and was functional both in vitro and in vivo. The different cofactor specificity between P. pastoris and C. guilliermondii xylose reductases might be due to the difference in the numbers of histidine residues and their locations between the two proteins. The recombinant was able to ferment xylose, and the maximum xylitol accumulation (7.8 g/l) was observed when the organism was grown under aerobic conditions. Received: 26 August 1997 / Received revision: 6 November 1997 / Accepted: 21 November 1997     

The genusCandida berkhout

The aim of the study was to verify the accuracy of the taxonomic classification of rough variants of the speciesCandida guilliermondii on the basis of comprehensive study of phenetic manifestation and to determine differences in cell wall structure with special reference to polysaccharides (1) According to their phenotype, the test strainsCandida guilliermondii (Cast.) Langeron et Guerra andCandida guilliermondii var.membranaefaciens Lodder et Kreger-Van Rij belong to the speciesCandida guilliermondii, whileCandida guilliermondii var.nitratophila Diddens et Lodder is phenotypically closer toCandida pelliculosa. (2) Observation of native and hydrolysed cell walls in the electron microscope showed no differences between the test strains. (3) The results of X-ray phase analysis of cell walls differentiatedCandida guilliermondii var.nitratophila from the other two, however. (4) Electron microscopy photomicrographs and diffractograms of cell walls indicated that, after 2% HCl extraction at 100 C, the cell walls contain chitin, which is isolated by further extraction with 30% HCl. After 3% NaOH hydrolysis the chitin diffractogram is not clear.     

Saccharomyces cerevisiae CCMI 885 secretes peptides that inhibit the growth of some non-Saccharomyces wine-related strains

The nature of the toxic compounds produced by Saccharomyces cerevisiae CCMI 885 that induce the early death of Hanseniaspora guilliermondii during mixed fermentations, as well as their ability to inhibit the growth of other non-Saccharomyces wine-related strains, was investigated. The killing effect of mixed supernatants towards H. guilliermondii was inactivated by protease treatments, thus revealing the proteinaceous nature of the toxic compounds. Analysis of the protein pattern of mixed supernatants on Tricine SDS-PAGE showed that this S. cerevisiae strain secretes peptides (<10 kDa), which were detected only when death of H. guilliermondii was already established. Death-inducing supernatants were ultrafiltrated by 10 and 2 kDa membranes, respectively, and the inhibitory effect of those permeates were tested in H. guilliermondii cultures. Results indicated that the (2–10) kDa protein fraction of those supernatants seemed to contain antimicrobial peptides active against H. guilliermondii. Thus, the (2–10) kDa protein fraction was concentrated and its inhibitory effect tested against strains of Kluyveromyces marxianus, Kluyveromyces thermotolerans, Torulaspora delbrueckii and H. guilliermondii. Under the growth conditions used for these tests, the (2–10) kDa protein fraction of S. cerevisiae CCMI 885 supernatants exhibited a fungistatic effect against all the strains and a fungicidal effect against K. marxianus.     

Optimization for high-level expression of the Pichia guilliermondii recombinant inulinase in Pichia pastoris and characterization of the recombinant inulinase

The inulinase gene cloned from the marine-derived yeast Pichia guilliermondii strain 1 was expressed in Pichia pastoris X-33 and the conditions for overexpression of the inulinase were optimized. After the optimization of the conditions for production of the recombinant inulinase, 286.8 ± 5.4 U/ml and 8873 ± 55.3 U/mg of the recombinanat inulinase in the supernatant of the culture of 2-l fermentor were attained at 120 h of the fermentation and fermentation efficiency was 13.04 μg ± 0.4 of protein/ml/d. The recombinant inulinase was purified and characterized. The molecular weight of the purified recombinant inulinase was 57.6 kDa, which was higher than that of the native iunlinase. The optimal pH and temperature of the purified recombinant inulinase were 6.0 and 60 °C, respectively. Other biochemical characteristics of the purified recombinant inulinase were the same as those of the native inulinase produced by the marine-derived P. guilliermondii strain 1. The purified recombinant inulinase also had high exoinulinase activity. Therefore, the recombinant inulinase may have highly potential applications in food and pharmaceutical industies.     

Molecular and physiological comparison of spoilage wine yeasts

Aims

Dekkera bruxellensis and Pichia guilliermondii are contaminating yeasts in wine due to the production of phenolic aromas. Although the degradation pathway of cinnamic acids, precursors of these phenolic compounds has been described in D. bruxellensis, no such pathway has been described in P. guilliermondii.

Methods and Results

A molecular and physiological characterization of 14 D. bruxellensis and 15 P. guilliermondii phenol‐producing strains was carried out. Both p‐coumarate decarboxylase (CD) and vinyl reductase (VR) activities, responsible for the production of volatile phenols, were quantified and the production of 4‐vinylphenol and 4‐ethylphenol were measured. All D. bruxellensis and some P. guilliermondii strains showed the two enzymatic activities, whilst 11 of the 15 strains of this latter species showed only CD activity and did not produce 4‐EP in the assay conditions. Furthermore, PCR products obtained with degenerated primers showed a low homology with the sequence of the gene for a phenyl acrylic acid decarboxylase activity described in Saccharomyces cerevisiae.

Conclusions

D. bruxellensis and P. guilliermondii may share a similar metabolic pathway for the degradation of cinnamic acids.

Significance and Impact of the Study

This is the first work that analyses the CD and VR activities in P. guilliermondii, and the results suggest that within this species, there are differences in the metabolization of cinnamic acids.     

Xylitol production by Candida species grown on a grass hydrolysate

Xylitol was produced by selected species of the yeast Candida after growth on a medium containing a hydrolysate of the North American perennial prairie grass big bluestem. The grass was hydrolysed by a combination of dilute acid and enzymatic treatments. After growth on the medium for 120 h at 30 °C, Candida tropicalis ATCC 750 produced a 1.4-fold higher level of xylitol than did C. tropicalis ATCC 20215 while biomass production by C. tropicalis ATCC 750 was 1.7-fold higher than Candida guilliermondii ATCC 20216. The xylitol yields observed for C. tropicalis ATCC 750, Candida mogii ATCC 18364 and C. guilliermondii ATCC 20216 were at least 1.4-fold higher than the yield observed for C. tropicalis ATCC 20215 after growth for 120 h at 30 °C.     

Deficiency in frataxin homologue YFH1 in the yeast Pichia guilliermondii leads to missregulation of iron acquisition and riboflavin biosynthesis and affects sulfate assimilation

Pichia guilliermondii is a representative of yeast species that overproduce riboflavin (vitamin B₂) in response to iron deprivation. P. guilliermondii YFH1 gene coding for frataxin homologue, eukaryotic mitochondrial protein involved in iron trafficking and storage, was identified and deleted. Constructed P. guilliermondii Δyfh1 mutant grew very poorly in a sucrose-containing synthetic medium supplemented with sulfate or sulfite as a sole sulfur source. Addition of sodium sulfide, glutathione, cysteine, methionine, N-acetyl-l-cysteine partially restored growth rate of the mutant suggesting that it is impaired in sulfate assimilation. Cellular iron content in Δyfh1 mutant was ~3–3.5 times higher as compared to the parental strain. It produced 50–70 times more riboflavin in iron sufficient synthetic media relative to the parental wild-type strain. Biomass yield of the mutant in the synthetic glutathione containing medium supplemented with glycerol as a sole carbon source was 1.4- and 2.6-fold increased as compared to sucrose and succinate containing media, respectively. Oxygen uptake of the Δyfh1 mutant on sucrose, glycerol or succinate, when compared to the parental strain, was decreased 5.5-, 1.7- and 1.5-fold, respectively. Substitution of sucrose or glycerol in the synthetic iron sufficient medium with succinate completely abolished riboflavin overproduction by the mutants. Deletion of the YFH1 gene caused hypersensitivity to hydrogen peroxide and exogenously added riboflavin and led to alterations in superoxide dismutase activities. Thus, deletion of the gene coding for yeast frataxin homologue has pleiotropic effect on metabolism in P. guilliermondii.     

Effect of inoculum level on xylitol production from rice straw hemicellulose hydrolysate byCandida guilliermondii

The effect of inoculum level on xylitol production byCandida guilliermondii was evaluated in a rice straw hemicellulose hydrolysate. High initial cell density did not show a positive effect in this bioconversion since increasing the initial cell density from 0.67 g L^–1 to 2.41 g L^–1 decreased both the rate of xylose utilization and xylitol accumulation. The maximum xylitol yield (0.71 g g^–1) and volumetric productivity (0.56 g L^–1 h^–1) were reached with an inoculum level of 0.9 g L^–1. These results show that under appropriate inoculum conditions rice straw hemicellulose hydrolysate can be converted into xylitol by the yeastC. guilliermondii with efficiency values as high as 77% of the theoretical maximum.     

L-Arabinose metabolism in Candida arabinofermentans PYCC 5603T and Pichia guilliermondii PYCC 3012: influence of sugar and oxygen on product formation

l-Arabinose utilization by the yeasts Candida arabinofermentans PYCC 5603^T and Pichia guilliermondii PYCC 3012 was investigated in aerobic batch cultures and compared, under similar conditions, to d-glucose and d-xylose metabolism. At high aeration levels, only biomass was formed from all the three sugars. When oxygen became limited, ethanol was produced from d-glucose, demonstrating a fermentative pathway in these yeasts. However, pentoses were essentially respired and, under oxygen limitation, the respective polyols accumulated—arabitol from l-arabinose and xylitol from d-xylose. Different l-arabinose concentrations and oxygen conditions were tested to better understand l-arabinose metabolism. P. guilliermondii PYCC 3012 excreted considerably more arabitol from l-arabinose (and also xylitol from d-xylose) than C. arabinofermentans PYCC 5603^T. In contrast to the latter, P. guilliermondii PYCC 3012 did not produce any traces of ethanol in complex l-arabinose (80 g/l) medium under oxygen-limited conditions. Neither sustained growth nor active metabolism was observed under anaerobiosis. This study demonstrates, for the first time, the oxygen dependence of metabolite and product formation in l-arabinose-assimilating yeasts.     

The Photodynamic Inactivation of the Yeast Candida guilliermondii in the Presence of Photodithazine

Photodithazine, a glucosamine salt of chlorin e ₆, is highly effective in sensitization of Candida guilliermondii cells to visible light. The sensitizing effect of photodithazine was found to be related to free or cell surface–bound molecules of this dye. Sodium azide (a singlet oxygen quencher) and propyl gallate (an inhibitor of lipid peroxidation) protected yeast cells from the photodithazine-enhanced photoinactivation.     

The shelf life and effectiveness of granular formulations of Metschnikowia pulcherrima and Pichia guilliermondii yeast isolates that control postharvest decay of citrus fruit

Our overall objectives were to prepare commercially acceptable formulations of the postharvest biological control yeasts, Metschnikowia pulcherrima and Pichia guilliermondii, which have a long storage life and to determine the effectiveness of these formulations to control postharvest green and blue moulds on citrus fruit. Yeasts, grown on a cane molasses-based medium, were combined with talc or kaolin carriers and various adjuvants and the viability of yeast in 12 formulations was determined over a 6 month period. Formulation no. 11, containing talc, sodium alginate, sucrose, and yeast extract, for both yeasts had a significantly higher viable yeast cell content over a 6 month storage period. Among the formulations, three formulations (formulations no. 5, 6, and 11) were selected for additional in vivo testing because they had higher levels of viability amongst yeast cell populations during storage and were easier to resuspend remained in suspension more easily. These formulations were tested on Satsuma mandarin and grapefruit to control green and blue moulds. Formulations no. 5, 6, and 11 for both yeasts effectively controlled green mould, while only formulation no. 11 with either yeast isolate M. pulcherrima (isolate M1/1) or P. guilliermondii (isolate P1/3) effectively controlled both blue and green moulds.     

Evolutionary Position of n-Alkane-assimilating Yeast Candida maltosa Shown by Nucleotide Sequence of Small-subunit Ribosomal RNA Gene

We clarified the evolutionary position of Candida maltosa, an n-alkane-assimilating yeast, by sequencing the nucleotides of the small-subunit ribosomal RNA gene. Phylogenetic analyses showed the close evolutionary relationships of C. maltosa with C. tropicalis, C. viswanathii, C. albicans, C. parapsilosis, and C. guilliermondii, forming a sub-group within this genus.     

Effects of yeast antagonist in combination with UV-C treatment on postharvest diseases of pear fruit

The yeast antagonist Candida guilliermondii and ultraviolet-C (UV-C) treatment were investigated for controlling infection following artificial inoculation with Penicillium expansum or Botrytis cinerea, or natural infection in pear fruit stored at 20°C. Applied separately, both C. guilliermondii and UV-C (5 kJ m⁻²) effectively inhibited decay caused by P. expansum or B. cinerea, and natural infection. The combination of C. guilliermondii and UV-C showed better control efficacy. Application of UV-C did not affect the growth of C. guilliermondii in pear fruit wounds, while UV-C induced a significant increase in the activities of chitinase, β-1,3-glucanase, catalase and peroxidase in pear fruit. The mechanism by which UV-C enhanced the biocontrol efficacy of C. guilliermondii may be related to the elicitation of defense responses in pear fruit. The combination of C. guilliermondii and UV-C radiation could be a promising method for the control of P. expansum and B. cinerea in pear fruit.     

Endophytic fungi from rhizomes of <Emphasis Type="Italic">Paris polyphylla</Emphasis> var. <Emphasis Type="Italic">yunnanensis</Emphasis>

About 63 fungal endophytic isolates were separated from rhizomes of Paris polyphylla var. yunnanensis, which is a traditional medicinal plant mainly distributed in China. The isolates were characterized and grouped based on the culture characteristics and the morphology of colony growth and conidia. Eleven representative ones were selected for further taxonomical identification. Five genera namely Fusarium, Gliocladiopsis, Gliomastix, Aspergillus and Cylindrocarpon were identified on the basis of their morphological characterizations. Of them, the most frequent genus was Fusarium (i.e. Ppf1, Ppf3 and Ppf14). Their ITS-rDNA sequences were compared with those available in the GeneBank databases to obtain the closest related species by BLAST analysis as well as to analyze their phylogenetic affiliation. The isolates were identified as Gliocladiopsis irregularis (Ppf2), Plectosphaerella cucumerina (Ppf4), Padospora sp. (Ppf6), Gliomastix murorum var. murorum (Ppf7), Aspergillus fumigatus (Ppf9), Pichia guilliermondii (Ppf10), Neonectria radicicola (anamorph: Cylindrocarpon) (Ppf12) and one uncultured mycorrhizal ascomycete (Ppf13) separately based on their morphological and molecular features. The molecular characters of the endophytic fungi were basically coincident with their morphology. The broad diversity and taxonomic spectrum were exhibited by the endophytic fungi from P. polyphylla var. yunnanensis.