Diaporthe diversity and pathogenicity revealed from a broad survey of grapevine diseases in Europe.

Species of Diaporthe are considered important plant pathogens, saprobes, and endophytes on a wide range of plant hosts. Several species are well-known on grapevines, either as agents of pre- or post-harvest infections, including Phomopsis cane and leaf spot, cane bleaching, swelling arm and trunk cankers. In this study we explore the occurrence, diversity and pathogenicity of Diaporthe spp. associated with Vitis vinifera in major grape production areas of Europe and Israel, focusing on nurseries and vineyards. Surveys were conducted in Croatia, Czech Republic, France, Hungary, Israel, Italy, Spain and the UK. A total of 175 Diaporthe strains were isolated from asymptomatic and symptomatic shoots, branches and trunks. A multi-locus phylogeny was established based on five genomic loci (ITS, tef1, cal, his3 and tub2), and the morphological characters of the isolates were determined. Preliminary pathogenicity tests were performed on green grapevine shoots with representative isolates. The most commonly isolated species were D. eres and D. ampelina. Four new Diaporthe species described here as D. bohemiae, D. celeris, D. hispaniae and D. hungariae were found associated with affected vines. Pathogenicity tests revealed D. baccae, D. celeris, D. hispaniae and D. hungariae as pathogens of grapevines. No symptoms were caused by D. bohemiae. This study represents the first report of D. ambigua and D. baccae on grapevines in Europe. The present study improves our understanding of the species associated with several disease symptoms on V. vinifera plants, and provides useful information for effective disease management.

First Report of Diplodia seriata as Causal Agent of Olive Dieback in Croatia.

In August 2010, a dieback of young olive (Olea europea L.) trees (cvs. Pendolino and Leccino) occurred in two orchards in Istria, Croatia. According to the producers, low temperatures during the winter severely damaged the plants and led to their decline. Distinctive symptoms, assumed fungal infection, were observed in internal tissue of stems and branches. Elongated brown necrosis, sometimes with black streaks, was visible under the bark, therefore Verticillium wilt was suspected. Of 1,086 trees in two orchards (4 ha), 165 (15%) showed symptoms. To isolate the causal agent, surface-sterilized wood chips of symptomatic tissue were placed on potato dextrose agar (PDA). Fungal colonies resembling Botryosphaeriaceae spp. grew from all wood fragments placed on PDA, and from these colonies, monohyphal isolates were obtained. For morphological identification, pycnidial formation was stimulated by growing the isolates on 2% water agar that included stems of plant species Foeniculum vulgare Mill. at room temperature under diffuse light. Pycnidia contained conidia that initially showed as hyaline, becoming light to dark brown as they matured, ovoid with truncated or rounded base and obtuse apex, aseptate, with wall moderately thick, externally smooth, roughened on the inner surface, and 22.8 to 23.5 × 9.6 to 10.5 µm. On the basis of these morphological characters, fungal species Diplodia seriata (teleomorph "Botryosphaeria" obtusa) was suspected (3). For molecular identification, four isolates (MN3, MN4, MN5, and MN6) were used for PCR to amplify the internal transcribed spacer (ITS) region and partial translation elongation factor 1-alpha (EF1-α) gene, using primers ITS4/ITS5 and EF1-728F/EF1-986R, respectively. Sequencing was performed with those amplified genes, then sequences were deposited in GenBank. Comparison of these sequences with GenBank sequences for referent D. seriata isolate CBS 112555 (AY259094 and AY573220) (3) showed 100% homology. On the basis of molecular data, the isolates were confirmed to be species D. seriata De Not. Pathogenicity tests were performed by inoculation of 2-year-old olive plants, six plants per tested cultivar (Pendolino and Leccino). For every cultivar, four plants were wounded and mycelium plugs from D. seriata cultures on PDA were placed on the wounds and sealed with Parafilm. Two control plants per tested cultivar were inoculated with sterile PDA plugs. After 2 months, six of eight inoculated plants wilted completely, and under the bark, brown necrosis was observed. D. seriata was constantly reisolated from the inoculated plants and fulfilled Koch's postulates and confirmed pathogenicity of D. seriata on olive as causal agent of olive dieback. Control plants showed no symptoms of the disease. This fungus has been recognized as the cause of fruit rot of olive (1) and branch canker or dieback in Spain (2). To our knowledge, this is the first report of D. seriata as a pathogen of olive in Croatia. Also, this is one of the first reports of D. seriata as the cause of olive dieback in the world, while Moral et al. (1,2) mostly reported it as the cause of olive fruit rot. Since the same symptoms of olive dieback were observed at other localities in Croatia, the disease could represent a serious threat, particularly for young olive orchards. References: (1) J. Moral et al. Plant Dis. 92:311, 2008. (2) J. Moral et al. Phytopathology 100:1340, 2010. (3) A. J. L. Phillips et al. Fungal Divers. 25:141, 2007.

Use of the yeast Hansenula polymorpha (Pichia angusta) to remove contaminating sugars from ethyl beta-D-fructofuranoside produced during sucrose ethanolysis catalysed by invertase.

Alkyl glycosides are interesting intermediates for the production of biodegradable surfactants. Synthesis of ethyl beta-d-fructofuranoside by invertase-catalysed ethanolysis of sucrose has been extensively reported in literature. However, this procedure yields mixtures of glucose, fructose, sucrose and ethyl beta-d-fructofuranoside. Purification of ethyl beta-d-fructofuranoside from such mixtures by chromatographic methods is laborious, difficult to scale up and requires organic solvents. The yeast Hansenula polymorpha grows rapidly on glucose, fructose and sucrose. Sucrose hydrolysis in this yeast is catalysed by an intracellular alpha-glucosidase ('maltase'); consequently, H. polymorpha should be unable to hydrolyse ethyl beta-d-fructofuranoside. Indeed, aerobic cultivation of H. polymorpha on sugar mixtures obtained by invertase-catalysed ethanolysis of sucrose resulted in the complete removal of contaminating sugars, leaving ethyl beta-d-fructofuranoside as the sole organic compound in culture supernatants. Pure ethyl beta-d-fructofuranoside was recovered from the supernatants by mixed-bed ion exchange chromatography with an 86% yield.

High-cell-density cultivation of yeasts on disaccharides in oxygen-limited batch cultures.

Many facultatively fermentative yeast species exhibit a "Kluyver effect": even under oxygen-limited growth conditions, certain disaccharides that support aerobic, respiratory growth are not fermented, even though the component monosaccharides are good fermentation substrates. This article investigates the applicability of this phenomenon for high-cell-density cultivation of yeasts. In glucose-grown batch cultures of Candida utilis CBS 621, the onset of oxygen limitation led to alcoholic fermentation and, consequently, a decrease of the biomass yield on sugar. In maltose-grown cultures, alcoholic fermentation did not occur and oxygen-limited growth resulted in high biomass concentrations (90 g dry weight L(-1) from 200 g L(-1) maltose monohydrate in a simple batch fermentation). It was subsequently investigated whether this principle could also be applied to Kluyveromyces species exhibiting a Kluyver effect for lactose. In oxygen-limited, glucose-grown chemostat cultures of K. wickerhamii CBS 2745, high ethanol concentrations and low biomass yields were observed. Conversely, ethanol was absent and biomass yields on sugar were high in oxygen-limited chemostat cultures grown on lactose. Batch cultures of K. wickerhamii grown on lactose exhibited the same growth characteristics as the maltose-grown C. utilis cultures: absence of ethanol formation and high biomass yields. Within the species K. marxianus, the occurrence of a Kluyver effect for lactose is known to be strain dependent. Thus, K. marxianus CBS 7894 could be grown to high biomass densities in lactose-grown batch cultures, whereas strain CBS 5795 produced ethanol after the onset of oxygen limitation and, consequently, yielded low amounts of biomass. Because the use of yeast strains exhibiting a Kluyver effect obviates the need for controlled substrate-feeding strategies to avoid oxygen limitation, such strains should be excellently suited for the production of biomass and growth-related products from low-cost disaccharide-containing feedstocks. (c) 1996 John Wiley & Sons, Inc.

Coordination of sucrose uptake and respiration in the yeast Debaryomyces yamadae.

Screening in batch cultures identified Debaryomyces yamadae as a yeast that exhibits the Kluyver effect for sucrose: this disaccharide can be respired but, even under oxygen-limited conditions, alcoholic fermentation of sucrose does not occur. Ethanol, glycerol and arabitol were the main fermentation products during oxygen-limited growth on glucose in chemostat cultures. None of these fermentation products were produced in oxygen-limited chemostat cultures grown on sucrose and the fraction of the sucrose that could not be respired remained unused in the culture medium. This absence of alcoholic fermentation was not due to repression of the key fermentative enzymes pyruvate decarboxylase and alcohol dehydrogenase. In contrast to some other yeasts that exhibit a Kluyver effect, D. yamadae did not exhibit a preference for ethanol in batch cultures grown on mixtures of ethanol and sucrose. Sucrose metabolism in D. yamadae involves intracellular hydrolysis by an alpha-glucosidase. Incubation of weakly buffered cell suspensions with sucrose led to a rapid transient alkalinization, indicating the presence of a sucrose-proton symport system. The apparent substrate saturation constant of the sucrose-uptake system was 0.2 mmol l-1. Sucrose-dependent alkalinization rates were much lower in samples from oxygen-limited cultures than in samples from aerobic cultures. Transient responses of D. yamadae to oxygen limitation were investigated by applying a sudden decrease in the oxygen feed to aerobic sugar-limited chemostat cultures. In glucose-grown cultures, this led to alcoholic fermentation and no significant accumulation of sugar occurred after the switch. In sucrose-limited cultures, sugar accumulation occurred instantaneously after the switch, and ethanol formation was virtually absent.(ABSTRACT TRUNCATED AT 250 WORDS)

Transient responses of Candida utilis to oxygen limitation: regulation of the Kluyver effect for maltose.

The facultatively fermentative yeast Candida utilis exhibits the Kluyver effect for maltose: this disaccharide is respired and assimilated but, in contrast to glucose, it cannot be fermented. To study the mechanism of the Kluyver effect, metabolic responses of C. utilis to a transition from aerobic, sugar-limited growth to oxygen-limited conditions were studied in chemostat cultures. Unexpectedly, the initial response of maltose-grown cultures to oxygen limitation was very similar to that of glucose-grown cultures. In both cases, alcoholic fermentation occurred after a lag phase of 1 h, during which glycerol, pyruvate and D-lactate were the main fermentation products. After ca. 10 h the behaviour of the maltose- and glucose-grown cultures diverged: ethanol disappeared from the maltose-grown cultures, whereas fermentation continued in steady-state, oxygen-limited cultures grown on glucose. The disappearance of alcoholic fermentation in oxygen-limited chemostat cultures growing on maltose was not due to a repression of the synthesis of pyruvate decarboxylase and alcohol dehydrogenase. The results demonstrate that the Kluyver effect for maltose in C. utilis does not reflect an intrinsic inability of this yeast to ferment maltose, but is caused by a regulatory phenomenon that affects a key enzyme in maltose metabolism, probably the maltose carrier. The observed kinetics indicate that this regulation occurs at the level of enzyme synthesis rather than via modification of existing enzyme activity.