Ecological basis of the interaction between Pseudozyma flocculosa and powdery mildew fungi

In this work, we sought to understand how glycolipid production and the availability of nutrients could explain the ecology of Pseudozyma flocculosa and its biocontrol activity. For this purpose, we compared the development of P. flocculosa to that of a close relative, the plant pathogen Ustilago maydis, under different environmental conditions. This approach was further supported by measuring the expression of cyp1, a pivotal gene in the synthesis of unique antifungal cellobiose lipids of both fungi. On healthy cucumber and tomato plants, the expression of cyp1 remained unchanged over time in P. flocculosa and was undetected in U. maydis. At the same time, green fluorescent protein (GFP) strains of both fungi showed only limited green fluorescence on control leaves. On powdery mildew-infected cucumber leaves, P. flocculosa induced a complete collapse of the pathogen colonies, but glycolipid production, as studied by cyp1 expression, was still comparable to that of controls. In complete contrast, cyp1 was upregulated nine times when P. flocculosa was applied to Botrytis cinerea-infected leaves, but the biocontrol fungus did not develop very well on the pathogen. Analysis of the possible nutrients that could stimulate the growth of P. flocculosa on powdery mildew structures revealed that the complex Zn/Mn played a key role in the interaction. Other related fungi such as U. maydis do not appear to have the same nutritional requirements and hence lack the ability to colonize powdery mildews. Whether production of antifungal glycolipids contributes to the release of nutrients from powdery mildew colonies is unclear, but the specificity of the biocontrol activity of P. flocculosa toward Erysiphales does appear to be more complex than simple antibiosis.     

The <Emphasis Type="Italic">Pseudozyma flocculosa</Emphasis> actin promoter allows the strong expression of a recombinant protein in the <Emphasis Type="Italic">Pseudozyma</Emphasis> species

Fungi belonging to the recently classified genus Pseudozyma possess some unique properties such as biocontrol activity, production of rare antimicrobial glycolipids and production of recombinant proteins. In this work, we report the first cloning of a promoter endogenous to the multi-faceted yeast-like Pseudozyma flocculosa, that of the actin gene. The promoter region lacked typical TATA or CAAT box but displayed three putative GC box and two CT-rich regions. As in other related basidiomycetes, only one copy of the actin gene was present in the genome of P. flocculosa. The activity of the actin promoter was compared to that of the HSP70 promoter from Ustilago maydis in two Pseudozyma species. In P. flocculosa, the actin promoter allowed the expression of a very high amount of GFP protein (27.8 mg g(-1) total protein) compared to those obtained with the HSP70 promoter in liquid culture. By contrast, the levels of GFP expression obtained in liquid culture were similar with the actin or the HSP70 promoter in Pseudozyma antarctica. A similar pattern of GFP expression was observed in solid culture. The cloning of this new promoter offers a unique genetic tool to further exploit and study the unusual properties of fungi from the Pseudozyma genus.     

Specificity and mode of action of the antifungal fatty acid cis-9-heptadecenoic acid produced by Pseudozyma flocculosa

cis-9-Heptadecenoic acid (CHDA), an antifungal fatty acid produced by the biocontrol agent Pseudozyma flocculosa, was studied for its effects on growth and/or spore germination in fungi. Inhibition of growth and/or germination varied considerably and revealed CHDA sensitivity groups within tested fungi. Analysis of lipid composition in these fungi demonstrated that sensitivity was related primarily to a low intrinsic sterol content and that a high level of unsaturation of phospholipid fatty acids was not as involved as hypothesized previously. Our data indicate that CHDA does not act directly with membrane sterols, nor is it utilized or otherwise modified in fungi. A structural mechanism of CHDA, consistent with the other related antifungal fatty acids produced by P. flocculosa, is proposed in light of its activity and specificity. The probable molecular events implicated in the sensitivity of fungi to CHDA are (i) partitioning of CHDA into fungal membranes; (ii) a variable elevation in fluidity dependent on the buffering capability (sterol content) in fungi; and (iii) higher membrane disorder causing conformational changes in membrane proteins, increased membrane permeability and, eventually, cytoplasmic disintegration.     

GFP technology for the study of biocontrol agents in tritrophic interactions: a case study with Pseudozyma flocculosa

GFP technology was applied to the biocontrol agent (BCA) Pseudozyma flocculosa to study its development and interactions at the tritrophic level plant-powdery mildew-BCA. Transformation experiments with GFP led to the production of a strongly fluorescent strain, Act-4, that displayed biocontrol traits typical of P. flocculosa WT. Following inundative applications, growth of P. flocculosa Act-4 was closely and almost exclusively associated with the colonies of the pathogen regardless of the powdery mildew species or the host plant tested. Development of P. flocculosa Act-4 on control leaves alone was extremely limited 24 h after its application and was typical of the epiphytic growth characterizing this type of yeast-like fungus. Based on the strong correlation between the colonization pattern of the different powdery mildew species tested and the presence of P. flocculosa Act-4, as determined by its fluorescence, it seems that growth of the BCA is dependant on the presence of powdery mildews. These results demonstrate that the GFP technology can be used to study plant-pathogen-BCA interactions and fulfill a wide array of purposes ranging from fundamental observations of the biocontrol behavior of a BCA to very applied ones serving some of the requirements for the registration of BCA's such as defining their environmental fate.     

Beta hydroxylation of glycolipids from Ustilago maydis and Pseudozyma flocculosa by an NADPH-dependent β-hydroxylase

Flocculosin and ustilagic acid (UA), two highly similar antifungal cellobiose lipids, are respectively produced by Pseudozyma flocculosa, a biocontrol agent, and Ustilago maydis, a plant pathogen. Both glycolipids contain a short-chain fatty acid hydroxylated at the β position but differ in the long fatty acid, which is hydroxylated at the α position in UA and at the β position in flocculosin. In both organisms, the biosynthesis genes are arranged in large clusters. The functions of most genes have already been characterized, but those of the P. flocculosa fhd1 gene and its homolog from U. maydis, uhd1, have remained undefined. The deduced amino acid sequences of these genes show homology to those of short-chain dehydrogenases and reductases (SDR). We disrupted the uhd1 gene in U. maydis and analyzed the secreted UA. uhd1 deletion strains produced UA lacking the β-hydroxyl group of the short-chain fatty acid. To analyze the function of P. flocculosa Fhd1, the corresponding gene was used to complement U. maydis Δuhd1 mutants. Fhd1 was able to restore wild-type UA production, indicating that Fhd1 is responsible for β hydroxylation of the flocculosin short-chain fatty acid. We also investigated a P. flocculosa homolog of the U. maydis long-chain fatty-acid alpha hydroxylase Ahd1. The P. flocculosa ahd1 gene, which does not reside in the flocculosin gene cluster, was introduced into U. maydis Δahd1 mutant strains. P. flocculosa Ahd1 neither complemented the U. maydis Δahd1 phenotype nor resulted in the production of β-hydroxylated UA. This suggests that P. flocculosa Ahd1 is not involved in flocculosin hydroxylation.     

Nutritional regulation and kinetics of flocculosin synthesis by Pseudozyma flocculosa

This study sought to identify the factors and conditions that affected production of the antifungal glycolipid flocculosin by the biocontrol agent Pseudozyma flocculosa. For this purpose, different parameters known or reported to influence glycolipid release in fungi were tested. Concentration of the start-up inoculum was found to play an important role in flocculosin production, as the optimal level increased productivity by as much as tenfold. Carbon availability and nitrogen source (i.e., organic vs inorganic) both had a direct influence on the metabolism of P. flocculosa, leading to flocculosin synthesis. In general, if conditions were conducive for production of the glycolipid, carbon availability appeared to be the only limiting factor. On the other hand, if yeast extract was supplied as nitrogen source, fungal biomass was immediately stimulated to the detriment of flocculosin synthesis. Unlike other reports of glycolipid release by yeast-like fungi, inorganic nitrogen starvation did not trigger production of flocculosin. The relationship between the factors influencing flocculosin production in vitro and the conditions affecting the release of the molecule by P. flocculosa in its natural habitat appears to be linked to the availability of a suitable and plentiful food source for the biocontrol agent.     

Insertional mutagenesis of a fungal biocontrol agent led to discovery of a rare cellobiose lipid with antifungal activity

Insertional mutagenesis was applied for the first time to a fungal biocontrol agent, Pseudozyma flocculosa, in an attempt to obtain mutants with altered antagonistic properties. Transformants were obtained via DNA-mediated transformation. Molecular analyses of the transformants revealed that multiple copies of the plasmid were integrated in tandem at one to many chromosomal loci. The transformants were screened for their biocontrol properties using standard bioassays, and the 160 tested transformants were classified into four groups: group I mutants (22 transformants) showed a stronger antagonistic effect than the wild type (WT) while those of group II (107 transformants) had a comparable antagonistic effect; group III mutants (17 transformants) had a decreased antagonistic effect relative to WT and group IV mutants (14 transformants) had lost their biocontrol properties. Culture extracts of the mutants (group IV) and WT were analyzed and compared for the presence of active metabolites which were then separated by solid-phase extraction and purified using conventional methods. Nuclear magnetic resonance experiments and analytical studies on a metabolite specifically produced by the WT revealed the presence of 2-(2',4'-diacetoxy-5'-carboxy-pentanoyl) octadecyl cellobioside (flocculosin), a novel glycolipid with strong antifungal properties; the production of this compound would account for the biocontrol activity of P. flocculosa.     

Establishment of a gene transfer system for Pseudozyma flocculosa, an antagonistic fungus of powdery mildew fungi

A reliable DNA-mediated transformation system has been developed for Pseudozyma flocculosa, a fungus that is antagonistic to powdery-mildew fungi. Plasmids harboring various selectable markers under the control of different promoters were tested. Molecular analyses demonstrated that successful transformation could be achieved using a plasmid that confers resistance to hygromycin B under the control of the Ustilago maydis hsp70 promoter and terminator sequences. On average, 1-40 (mean = 20) transformants were obtained per 10 microg of linearized DNA per 10(8) protoplasts. Southern analysis of the transformants revealed that, in each case, the vector had integrated in multiple tandem copies into the genome of P. flocculosa, and that integration events were random. Pulsed-field gel electrophoresis was employed to separate the genome of P. flocculosa into at least 11 chromosomes with sizes ranging from 0.55 Mb to 2.9 Mb. Hybridization with the plasmid indicated that integration of vector DNA had occurred in one to several chromosomes depending on the transformant examined.     

Antifungal activity of extracellular metabolites produced by Sporothrix flocculosa

The antifungal properties of extracellular compounds produced by the epiphytic fungus Sporothrix flocculosa were bioassayed against phytopathogenie fungi on the basis of inhibition of spore germination, and mycelial growth and induction of cellular leakage. Following incubation in stationary culture, S. flocculosa released antifungal metabolites into the culture medium which were extractable with méthylene chloride. When separated by thin layer chromatography, extracted metabolites yielded a compound(s) at R_f0.65 which inhibited development of Cladosporium cucumerinum and several other phytopathogenic fungi. Treatment of Botrytis cinerea and Fusarium oxysporum f.sp. radicis‐lycopersici (FORL) with the same compound(s) greatly reduced spore germination and biomass growth of both fungi. Additionally, both B. cinerea and FORL leaked electrolytes and proteins when grown in presence of the metabolites. Observations under electron microscopy revealed that FORL reacted to the presence of S. flocculosa metabolites by retraction of the plasmalemma and rapid disintegration of the cytoplasm. These reactions were similar to the ones induced by conidia of S. flocculosa when applied on powdery mildew fungi. These results provide strong evidence of the production of antifungal compounds in vivo and of their role in the antagonistic properties of S. flocculosa.     

The potential of Pseudozyma yeastlike epiphytes for the production of heterologous recombinant proteins

Although Basidiomycetes represent the most evolved class of fungi, they have been neglected with regard to recombinant gene expression. In this work, basidiomycetous yeasts belonging to Pseudozyma spp. were studied with respect to their amenability to heterologous protein production. Single plasmid or cotransformation experiments routinely afforded 100 to 200 independent transformants for the two tested species of Pseudozyma. Green fluorescent protein (GFP) was expressed in the correctly folded conformation, as demonstrated by fluorescence microscopy, and hen egg white lysozyme (HEWL) was expressed in its active form, as revealed by its lytic activity on Micrococcus lysodeikticus cells. Protease analysis established that Pseudozyma spp. contained equivalent or less extracellular protease activity than yeasts and far less protease activity than ascomycetous filamentous fungi in similar culture conditions. This proteolytic activity was inhibited by over 97% with a combination of PMSF and Pepstatin A. N-glycosylation patterns of native Pseudozyma flocculosa secreted proteins were comprised of one or a few short glycan chains that possess a classic eukaryotic structure typical of higher fungi and animal cells. This is the first report of a Basidiomycete that possesses multiple intrinsic characteristics necessary for use as a heterologous gene expression system.     

Safety and regulation of yeasts used for biocontrol or biopreservation in the food or feed chain

Yeasts have been important components of spontaneous fermentations in food and beverage processing for millennia. More recently, the potential of utilising antagonistic yeasts, e.g. Pichia anomala and Candida spp., for post-harvest biological control of spoilage fungi during storage of plant-derived produce (‘biopreservation’) has been clearly demonstrated. Although some yeast species are among the safest microorganisms known, several have been reported in opportunistic infections in humans, including P. anomala and bakers’ yeast, Saccharomyces cerevisiae. More research is needed about the dominant pathogenicity and virulence factors in opportunistic yeasts, and whether increased utilisation of biopreservative yeasts in general could contribute to an increased prevalence of yeast infections. The regulatory situation for yeasts used in post-harvest biocontrol is complex and the few products that have reached the market are mainly registered as biological pesticides. The qualified presumption of safety (QPS) approach to safety assessments of microorganisms intentionally added to food or feed, recently launched by the European Food Safety Authority, can lead to more efficient evaluations of new products containing microbial species with a sufficient body of knowledge or long-term experience on their safety. P. anomala is one of several yeast species that have been given QPS status, although the status is restricted to use of this yeast for enzyme and metabolite production purposes. With regard to authorisation of new biopreservative yeasts, we recommend that the possibility to regulate microorganisms for food biopreservation as food additives be considered.     

Identity and biodegradative abilities of yeasts isolated from plants growing in an arid climate

Plants harvested in the Canary Islands Lanzarote and Fuerteventura were analyzed for the yeasts inhabiting their surface. Half of the isolates (22 out of 44) were identified as Debaryomyces hansenii. Black ascomycetes, viz. Hortaea werneckii and two Hormonema species were represented by 7 strains. Basidiomycetous yeasts, viz. Cryptococcus sp. (8 strains), Rhodotorula sp. (5 strains), Cerinosterus cyanescens (1 strain) and Pseudozyma sp. (1 strain) constituted a minority of 33%. Thirty strains were screened for their ability to assimilate various plant constituents including lipids of the cuticle and the cell membrane, hemicelluloses, nitrogenous compounds (protein, nucleic acids, amino acids) and benzene compounds. All strains were able to assimilate or to hydrolyze lipids, lecithin included. Many strains of D. hansenii, H. dematioides, H. werneckii, C. cyanescens, Cr. laurentii, Pseudozyma sp. and Rh. glutinis were proteolytic. Hemicelluloses like xylan and pectin were assimilated by black ascomycetous yeasts, Cryptococcus sp., Pseudozyma sp. and Rh. glutinis. Ferulic and hydroxycinnamic acids, gallic and tannic acids were assimilated by some strains of H. dematioides, C. cyanescens, Pseudozyma sp. and Rhodotorula sp.     

Usefulness of heterologous promoters in the Pseudozyma flocculosa gene expression system

The basidiomycetous fungus Pseudozyma flocculosa represents a promising new host for the expression of complex recombinant proteins. Two novel heterologous promoter sequences, the Ustilago maydis glyceraldehyde-3-phosphate dehydrogenase (GPD) and Pseudozyma tsukubaensis alpha-glucosidase promoters, were tested for their ability to provide expression in P. flocculosa. In liquid medium, these two promoters produced lower levels of intracellular green fluorescent protein (GFP) as compared to the U. maydis hsp70 promoter. However, GPD and alpha-glucosidase sequences behaved as constitutive promoters whereas the hsp70 promoter appeared to be morphology-dependent. When using the hsp70 promoter, the expression of GFP increased proportionally to the concentration of hygromycin in the culture medium, indicating possible induction of the promoter by the antibiotic. Optimal solid-state culture conditions were designed for high throughput screening of hygromycin-resistant transformants with the hsp70 promoter in P. flocculosa.     

Production of different types of mannosylerythritol lipids as biosurfactants by the newly isolated yeast strains belonging to the genus Pseudozyma

Mannosylerythritol lipids (MEL), which are abundantly secreted by yeasts, are one of the most promising biosurfactants known. To obtain various types of MEL and to attain a broad range of applications for them, screening of novel producers was undertaken. Thirteen strains of yeasts were successfully isolated as potential MEL producers; they showed high production yields of MEL of around 20 g l(-1) from 40 g l(-1) of soybean oil. Based on the taxonomical study, all the strains were classified to be the genus Pseudozyma. It is interesting to note that they were categorized into three groups according to their production patterns of MEL. The first group, which included 11 strains taxonomically closely related to high-level MEL producers such as Pseudozyma antarctica and Pseudozyma aphidis, mainly produced 4-O-[(4',6'-di-O-acetyl-2',3'-di-O-alkanoyl)-beta-D-mannopyranosyl]-meso-erythritol (MEL-A) together with 4-O-[(6'-mono-O-acetyl-2',3'-di-O-alkanoyl)-beta-D-mannopyranosyl]-meso-erythritol (MEL-B) and 4-O-[(4'-mono-O-acetyl-2',3'-di-O-alkanoyl)-beta-D-mannopyranosyl]-meso-erythritol (MEL-C) as the minor components. The second group of one strain, which was related to Pseudozyma tsukubaensis, predominantly produced MEL-B. The third group of one strain, which was closely related to Pseudozyma hubeiensis, mainly produced MEL-C; this is the first observation of the efficient production of MEL-C from soybean oil. Moreover, the major fatty acids of the obtained MEL-C were C(6), C(12), and C(16) acids, and were considerably different from those of the other MEL hitherto reported. The biosynthetic manner for MEL is thus likely to significantly vary among the Pseudozyma strains; the newly isolated strains would enable us to attain a large-scale production of MEL and to obtain various types of MEL with different hydrophobic structures.     

Pathogenicity of the Fungus Verticillium lecanii to Aphids and Powdery Mildew

Aspects of the host specificity and pathogenicity of the hyphomycete, Verticillium lecanii , were investigated under laboratory conditions. DiVerences were observed in the pathogenicities of three strains of V. lecanii (DAOM 198499, DAOM 216596 and Vertalec) to the potato aphid Macrosiphum euphorbiae and Sphaerotheca fuliginea , the causal agent of cucumber powdery mildew. The estimated median lethal concentration required to achieve 50% mortality (LC ), 50 median lethal time leading to 50% mortality (LT ) and aphid net reproductive rate ( R ) 50 0 indicated that Vertalec and strain 198499 were more virulent to aphids than strain 216596. The estimated median colonization time for 50% of fungal colonies (CT ) showed that strain 50 198499 was the best antagonist of cucumber powdery mildew. Further comparison suggested that the mean pathogenicity of V. lecanii strain 198499 to cucumber powdery mildew was almost equivalent to that of Sporothrix flocculosa , a biological control agent of greenhouse fungal pathogens. These observations provide convincing experimental evidence that V. lecanii is biologically active against both arthropods and fungi. The potential of using V. lecanii strain 198499 in biological control is discussed.     

Molecular mechanisms of peroxisome biogenesis in yeasts

Peroxisomes contain oxidases generating hydrogen peroxide, and catalase degrading this toxic compound. Another characteristic function of each eukaryotic peroxisome, from yeast to man, is fatty acid beta-oxidation. However, in peroxisomes a variety of other metabolic pathways are located. In fungi, peroxisomes contain enzymes involved in catabolism of unusual carbon and nitrogen sources (methanol, purines, D-amino acids, pipecolynic acid, sarcosine, glycolate, spermidine etc) as well as biosynthesis of lysine in yeasts and penicillin in mycelial fungi. Impairment of peroxisomal structure and functions causes many human disorders. The similar defects have been identified in yeast mutants defective in peroxisomal biogenesis. Peroxisomal biogenesis is actively studied during last two decades using uni- and multicellular model systems. It was observed that many aspects of peroxisomal biogenesis and proteins involved in this process display striking similarity between all eukaryotes, from yeasts to humans. Yeast is a convenient model system for this kind of research. Current review summarizes data on molecular events of peroxisomal biogenesis, functions of peroxine proteins, import of peroxisomal matrix and membrane proteins and on mechanisms of peroxisomedivision and inheritance.     

Hydroxy long-chain fatty acids in fungi

Hydroxy long-chain fatty acids occur widely in animals and plants and have important physiological activities in these eukaryotes. There are indications that these compounds are also common and important in fungi. The occurrence of hydroxy-polyunsaturated fatty acids (hydroxy-PUFAs) is of biotechnological importance, because these compounds are potentially high-value lipid products with medical applications. This review pays particular attention to the production of hydroxy-PUFAs by yeasts and other fungi. Hydroxy-PUFAs derived from lipoxygenase activity appear to be present in most fungi, while hydroxy-PUFAs from cyclooxygenase activity (i.e. prostaglandins) have mainly been implicated in the Oomycota and in yeasts from the genus Dipodascopsis. The occurrence of other hydroxy long-chain fatty acids in fungi is also discussed briefly; these include hydroxy fatty acids that are generally associated with cytochrome P-450 monooxygenase activity (i.e. terminal and sub-terminal hydroxy acids and diols derived from the corresponding epoxides) as well as 2-hydroxy-fatty acids and 3-hydroxy-fatty acids.The authors are with the Department of Microbiology and Biochemistry, University of the Orange Free State, P.O. Box 339, Bloemfontein, 9300, South Africa     

Effect of foliar disease on the epiphytic yeast communities of creeping bentgrass and tall fescue

The effect of mechanical wounding or foliar diseases caused by Sclerotinia homoeocarpa or Rhizoctonia solani on the epiphytic yeast communities on creeping bentgrass and tall fescue were determined by leaf washing and dilution plating. Total yeast communities on healthy bentgrass and tall fescue leaves ranged from 7.9 x 103 to 1.4 x 105 CFU.cm-2 and from 2.4 x 103 to 1.6 x 104 CFU.cm-2, respectively. Mechanically wounded leaves (1 of 2 trials) and leaves with disease lesions (11 of 12 trials) supported significantly larger communities of phylloplane yeasts. Total yeast communities on S. homoeocarpa infected or R. solani infected bentgrass leaves were 3.6-10.2 times and 6.2-6.4 times larger, respectively, than the communities on healthy leaves. In general, healthy and diseased bentgrass leaves supported larger yeast communities than healthy or diseased tall fescue leaves. We categorized the majority of yeasts as white-pigmented species, including Cryptococcus laurentii, Cryptococcus flavus, Pseudozyma antarctica, Pseudozyma aphidis, and Pseudozyma parantarctica. The percentage of pink yeasts in the total yeast community ranged from 2.6% to 9.9% on healthy leaves and increased to 32.0%-44.7% on S. homoeocarpa infected leaves. Pink-pigmented yeasts included Rhodotorula glutinis, Rhodotorula mucilaginosa, Sakaguchia dacryoidea, and Sporidiobolus pararoseus. Foliar disease significantly affected community size and composition of epiphytic yeasts on bentgrass and tall fescue.     

Molecular mechanisms of peroxisome biogenesis in yeasts

Peroxisomes contain oxidases, which generate hydrogen peroxide, and catalase, which degrades this toxic compound. Another characteristic function of each eukaryotic peroxisome, from yeast to man, is fatty acid β-oxidation. However, a variety of other metabolic pathways are also located in peroxisomes. In fungi, peroxisomes contain enzymes involved in catabolism of unusual carbon and nitrogen sources (methanol, purines, D-amino acids, pipecolynic acid, sarcosine, glycolate, spermidine, etc.), as well as biosynthesis of lysine in yeasts and penicillin in mycelial fungi. Impairment of the peroxisome structure and functions causes many human disorders. Similar defects were identified in yeast mutants defective in peroxisome biogenesis. Peroxisome biogenesis has been actively studied using unicellular and multicellular model systems over the last two decades. It was observed that many aspects of peroxisome biogenesis and proteins involved in the process display striking similarity among all eukaryotes from yeasts to humans. Yeasts provide a convenient model system for this kind of research. The review summarizes the data on the molecular events of peroxisome biogenesis, the functions of peroxine proteins, the import of peroxisomal matrix and membrane proteins, and the mechanisms of peroxisome division and inheritance.