The effects of external Mn2+ concentration on hyphal morphology and citric acid production are mediated primarily by the NRAMP-family transporter DmtA in Aspergillus niger.

BACKGROUND: Citric acid, a commodity product of industrial biotechnology, is produced by fermentation of the filamentous fungus Aspergillus niger. A requirement for high-yield citric acid production is keeping the concentration of Mn2+ ions in the medium at or below 5 µg L-1. Understanding manganese metabolism in A. niger is therefore of critical importance to citric acid production. To this end, we investigated transport of Mn2+ ions in A. niger NRRL2270. RESULTS: we identified an A. niger gene (dmtA; NRRL3_07789), predicted to encode a transmembrane protein, with high sequence identity to the yeast manganese transporters Smf1p and Smf2p. Deletion of dmtA in A. niger eliminated the intake of Mn2+ at low (5 µg L-1) external Mn2+ concentration, and reduced the intake of Mn2+ at high (> 100 µg L-1) external Mn2+ concentration. Compared to the parent strain, overexpression of dmtA increased Mn2+ intake at both low and high external Mn2+ concentrations. Cultivation of the parent strain under Mn2+ ions limitation conditions (5 µg L-1) reduced germination and led to the formation of stubby, swollen hyphae that formed compact pellets. Deletion of dmtA caused defects in germination and hyphal morphology even in the presence of 100 µg L-1 Mn2+, while overexpression of dmtA led to enhanced germination and normal hyphal morphology at limiting Mn2+ concentration. Growth of both the parent and the deletion strains under citric acid producing conditions resulted in molar yields (Yp/s) of citric acid of > 0.8, although the deletion strain produced ~ 30% less biomass. This yield was reduced only by 20% in the presence of 100 µg L-1 Mn2+, whereas production by the parent strain was reduced by 60%. The Yp/s of the overexpressing strain was 17% of that of the parent strain, irrespective of the concentrations of external Mn2+. CONCLUSIONS: Our results demonstrate that dmtA is physiologically important in the transport of Mn2+ ions in A. niger, and manipulation of its expression modulates citric acid overflow.

l-Arabinose induces d-galactose catabolism via the Leloir pathway in Aspergillus nidulans.

l-Arabinose and d-galactose are the principal constituents of l-arabinogalactan, and also co-occur in other hemicelluloses and pectins. In this work we hypothesized that similar to the induction of relevant glycoside hydrolases by monomers liberated from these plant heteropolymers, their respective catabolisms in saprophytic and phytopathogenic fungi may respond to the presence of the other sugar to promote synergistic use of the complex growth substrate. We showed that these two sugars are indeed consumed simultaneously by Aspergillus nidulans, while l-arabinose is utilised faster in the presence than in the absence of d-galactose. Furthermore, the first two genes of the Leloir pathway for d-galactose catabolism - encoding d-galactose 1-epimerase and galactokinase - are induced more rapidly by l-arabinose than by d-galactose eventhough deletion mutants thereof grow as well as a wild type strain on the pentose. d-Galactose 1-epimerase is hyperinduced by l-arabinose, d-xylose and l-arabitol but not by xylitol. The results suggest that in A. nidulans, l-arabinose and d-xylose - both requiring NADPH for their catabolisation - actively promote the enzyme infrastructure necessary to convert ß-d-galactopyranose via the Leloir pathway with its α-anomer specific enzymes, into ß-d-glucose-6-phosphate (the starting substrate of the oxidative part of the pentose phosphate pathway) even in the absence of d-galactose.

Citric acid and itaconic acid accumulation: variations of the same story?

Citric acid production by Aspergillus niger and itaconic acid production by Aspergillus terreus are two major examples of technical scale fungal fermentations based on metabolic overflow of primary metabolism. Both organic acids are formed by the same metabolic pathway, but whereas citric acid is the end product in A. niger, A. terreus performs two additional enzymatic steps leading to itaconic acid. Despite of this high similarity, the optimization of the production process and the mechanism and regulation of overflow of these two acids has mostly been investigated independently, thereby ignoring respective knowledge from the other. In this review, we will highlight where the similarities and the real differences of these two processes occur, which involves various aspects of medium composition, metabolic regulation and compartmentation, transcriptional regulation, and gene evolution. These comparative data may facilitate further investigations of citric acid and itaconic acid accumulation and may contribute to improvements in their industrial production.

A mechanism for a single nucleotide intron shift.

Spliceosomal introns can occupy nearby rather than identical positions in orthologous genes (intron sliding or shifting). Stwintrons are complex intervening sequences, where an 'internal' intron interrupts one of the sequences essential for splicing, generating after its excision, a newly formed canonical intron defined as 'external'. In one experimentally demonstrated configuration, two alternatively excised internal introns, overlapping by one G, disrupt respectively the donor and the acceptor sequence of an external intron, leading to mRNAs encoding identical proteins. In a gene encoding a DHA1 antiporter in Pezizomycotina, we find a variety of predicted intron configurations interrupting the DNA stretch encoding a conserved peptidic sequence. Some sport a stwintron where the internal intron interrupts the donor of the external intron (experimentally confirmed for Aspergillus nidulans). In others, we found and demonstrate (for Trichoderma reesei) alternative, overlapping internal introns. Discordant canonical introns, one nt apart, are present in yet other species, exactly as predicted by the alternative loss of either of the internal introns at the DNA level from an alternatively spliced stwintron. An evolutionary pathway of 1 nt intron shift, involving an alternatively spliced stwintron intermediate is proposed on the basis of the experimental and genomic data presented.

High oxygen tension increases itaconic acid accumulation, glucose consumption, and the expression and activity of alternative oxidase in Aspergillus terreus.

Itaconic acid is a five-carbon dicarboxylic acid with an unsaturated alkene bond, frequently used as a building block for the industrial production of a variety of synthetic polymers. It is also one of the major products of fungal "overflow metabolism" which can be produced in submerged fermentations of the filamentous fungus Aspergillus terreus. At the present, molar yields of itaconate are lower than those obtained in citric acid production in Aspergillus niger. Here, we have studied the possibility that the yield may be limited by the oxygen supply during fermentation and hence tested the effect of the dissolved oxygen concentration on the itaconic acid formation rate and yield in lab-scale bioreactors. The data show that a dissolved oxygen concentration of 2% saturation was sufficient for maximal biomass formation. Raising it to 30% saturation had no effect on biomass formation or the growth rate, but the itaconate yield augmented substantially from 0.53 to 0.85 mol itaconate/mol glucose. Furthermore, the volumetric and specific rates of itaconic acid formation ameliorated by as much as 150% concurrent with faster glucose consumption, shortening the fermentation time by 48 h. Further increasing the dissolved oxygen concentration over 30% saturation had no effect. Moreover, we show that this increase in itaconic acid production coincides with an increase in alternative respiration, circumventing the formation of surplus ATP by the cytochrome electron transport chain, as well as with increased levels of alternative oxidase transcript. We conclude that high(er) itaconic acid accumulation requires a dissolved oxygen concentration that is much higher than that needed for maximal biomass formation, and postulate that the induction of alternative respiration allows the necessary NADH reoxidation ratio without surplus ATP production to increase the glucose consumption and the flux through overflow metabolism.

Characterization of a second physiologically relevant lactose permease gene (lacpB) in Aspergillus nidulans.

In Aspergillus nidulans, uptake rather than hydrolysis is the rate-limiting step of lactose catabolism. Deletion of the lactose permease A-encoding gene (lacpA) reduces the growth rate on lactose, while its overexpression enables faster growth than wild-type strains are capable of. We have identified a second physiologically relevant lactose transporter, LacpB. Glycerol-grown mycelia from mutants deleted for lacpB appear to take up only minute amounts of lactose during the first 60 h after a medium transfer, while mycelia of double lacpA/lacpB-deletant strains are unable to produce new biomass from lactose. Although transcription of both lacp genes was strongly induced by lactose, their inducer profiles differ markedly. lacpA but not lacpB expression was high in d-galactose cultures. However, lacpB responded strongly also to ß-linked glucopyranose dimers cellobiose and sophorose, while these inducers of the cellulolytic system did not provoke any lacpA response. Nevertheless, lacpB transcript was induced to higher levels on cellobiose in strains that lack the lacpA gene than in a wild-type background. Indeed, cellobiose uptake was faster and biomass formation accelerated in lacpA deletants. In contrast, in lacpB knockout strains, growth rate and cellobiose uptake were considerably reduced relative to wild-type, indicating that the cellulose and lactose catabolic systems employ common elements. Nevertheless, our permease mutants still grew on cellobiose, which suggests that its uptake in A. nidulans prominently involves hitherto unknown transport systems.

D-galactose catabolism in Penicillium chrysogenum: Expression analysis of the structural genes of the Leloir pathway.

In this study, we analyzed the expression of the structural genes encoding the five enzymes comprising the Leloir pathway of D-galactose catabolism in the industrial cell factory Penicillium chrysogenum on various carbon sources. The genome of P. chrysogenum contains a putative galactokinase gene at the annotated locus Pc13g10140, the product of which shows strong structural similarity to yeast galactokinase that was expressed on lactose and D-galactose only. The expression profile of the galactose-1-phosphate uridylyl transferase gene at annotated locus Pc15g00140 was essentially similar to that of galactokinase. This is in contrast to the results from other fungi such as Aspergillus nidulans, Trichoderma reesei and A. niger, where the ortholog galactokinase and galactose-1-phosphate uridylyl transferase genes were constitutively expressed. As for the UDP-galactose-4-epimerase encoding gene, five candidates were identified. We could not detect Pc16g12790, Pc21g12170 and Pc20g06140 expression on any of the carbon sources tested, while for the other two loci (Pc21g10370 and Pc18g01080) transcripts were clearly observed under all tested conditions. Like the 4-epimerase specified at locus Pc21g10370, the other two structural Leloir pathway genes - UDP-glucose pyrophosphorylase (Pc21g12790) and phosphoglucomutase (Pc18g01390) - were expressed constitutively at high levels as can be expected from their indispensable function in fungal cell wall formation.

Alternatively spliced, spliceosomal twin introns in Helminthosporium solani.

Spliceosomal twin introns, "stwintrons", have been defined as complex intervening sequences that carry a second intron ("internal intron") interrupting one of the conserved sequence domains necessary for their correct splicing via consecutive excision events. Previously, we have described and experimentally verified stwintrons in species of Sordariomycetes, where an "internal intron" interrupted the donor sequence of an "external intron". Here we describe and experimentally verify two novel stwintrons of the potato pathogen Helminthosporium solani. One instance involves alternative splicing of an internal intron interrupting the donor domain of an external intron and a second one interrupting the acceptor domain of an overlapping external intron, both events leading to identical mature mRNAs. In the second case, an internal intron interrupts the donor domain of the external intron, while an alternatively spliced intron leads to an mRNA carrying a premature chain termination codon. We thus extend the stwintron concept to the acceptor domain and establish a link of the occurrence of stwintrons with that of alternative splicing.

A deficiency of manganese ions in the presence of high sugar concentrations is the critical parameter for achieving high yields of itaconic acid by Aspergillus terreus.

Itaconic acid (IA), an unsaturated dicarboxylic acid with a high potential as a platform for chemicals derived from sugars, is industrially produced by large-scale submerged fermentation by Aspergillus terreus. Although the biochemical pathway and the physiology leading to IA is almost the same as that leading to citric acid production in Aspergillus niger, published data for the volumetric (g L(-1)) and the specific yield (mol/mol carbon source) of IA are significantly lower than for citric acid. Citric acid is known to accumulate to high levels only when a number of nutritional parameters are carefully adjusted, of which the concentration of the carbon source and that of manganese ions in the medium are particularly important. We have therefore investigated whether a variation in these two parameters may enhance IA production and yield by A. terreus. We show that manganese ion concentrations < 3 ppb are necessary to obtain highest yields. Highest yields were also dependent on the concentration of the carbon source (D-glucose), and highest yields (0.9) were only obtained at concentrations of 12-20 % (w/v), thus allowing the accumulation of up to 130 g L(-1) IA. These findings perfectly mirror those obtained when these parameters are varied in citric acid production by A. niger, thus showing that the physiology of both processes is widely identical. Consequently, applying the fermentation technology established for citric acid production by A. niger citric acid production to A. terreus should lead to high yields of IA, too.

The transcriptome of lae1 mutants of Trichoderma reesei cultivated at constant growth rates reveals new targets of LAE1 function.

BACKGROUND: The putative methyltransferase LaeA is a global regulator that affects the expression of multiple secondary metabolite gene clusters in several fungi. In Trichoderma reesei, its ortholog LAE1 appears to predominantly regulate genes involved in increasing competitive fitness in its environment, including expression of cellulases and polysaccharide hydrolases. A drawback in all studies related to LaeA/LAE1 function so far, however, is that the respective loss-of-function and overexpressing mutants display different growth rates. Thus some of the properties attributed to LaeA/LAE1 could be simply due to changes of the growth rate. RESULTS: We cultivated T. reesei, a Δlae1 mutant and a lae1-overexpressing strain in chemostats on glucose at two different growth rates (0.075 and 0.020 h-1) which resemble growth rates at repressing and derepressing conditions, respectively. Under these conditions, the effect of modulating LAE1 expression was mainly visible in the Δlae1 mutant, whereas the overexpressing strain showed little differences to the parent strain. The effect on the expression of some gene categories identified earlier (polyketide synthases, heterokaryon incompatibility proteins, PTH11-receptors) was confirmed, but in addition GCN5-N-acetyltransferases, amino acid permeases and flavin monooxygenases were identified as so far unknown major targets of LAE1 action. LAE1 was also shown to interfere with the regulation of expression of several genes by the growth rate. About a tenth of the genes differentially expressed in the Δlae1 mutant under either growth condition were found to be clustered in the genome, but no specific gene group was associated with this phenomenon. CONCLUSIONS: Our data show that - using T. reesei LAE1 as a model - the investigation of transcriptome in regulatory mutants at constant growth rates leads to new insights into the physiological roles of the respective regulator.

Transcriptomics identify the triggering of citrate export as the key event caused by manganese deficiency in Aspergillus niger.

For over a century, the filamentous Ascomycete fungus Aspergillus niger has played a pivotal role in the industrial production of citric acid. A critical fermentation parameter that sustains high-yield citric acid accumulation is the suboptimal concentration of manganese(II) ions in the culture broth at the early stages of the process. However, the requirement for this deficiency has not been investigated on a functional genomics level. In this study, we compared the transcriptome of the citric acid hyper-producer A. niger NRRL2270 strain grown under citric acid-producing conditions in 6-L scale bioreactors at Mn2+ ion-deficient (5 ppb) and Mn2+ ion-sufficient (100 ppb) conditions at three early time points of cultivation. Of the 11,846 genes in the genome, 963 genes (8.1% of the total) were identified as significantly differentially expressed under these conditions. Disproportionately high number of differentially regulated genes encode predicted extracellular and membrane proteins. The most abundant gene group that was upregulated in Mn2+ ion deficiency condition encodes enzymes acting on polysaccharides. In contrast, six clusters of genes encoding secondary metabolites showed downregulation under manganese deficiency. Mn2+ deficiency also triggers upregulation of the cexA gene, which encodes the citrate exporter. We provide functional evidence that the upregulation of cexA is caused by the intracellular accumulation of citrate or acetyl-CoA and is a major factor in triggering citrate overflow. IMPORTANCE: Citric acid is produced on industrial scale by batch fermentation of the filamentous fungus Aspergillus niger. High-yield citric acid production requires a low (<5 ppb) manganese(II) ion concentration in the culture broth. However, the requirement for this deficiency has not been investigated on a functional genomics level. Here, we compared the transcriptome of a citric acid hyper-producer A. niger strain grown under citric acid-producing conditions in 6-L scale bioreactors at Mn2+ ion-deficient (5 ppb) and Mn2+ ion-sufficient (100 ppb) conditions at three early time points of cultivation. We observed that Mn2+ deficiency triggers an upregulation of the citrate exporter gene cexA and provides functional evidence that this event is responsible for citrate overflow. In addition to the industrial relevance, this is the first study that examined the role of Mn2+ ion deficiency in a heterotrophic eukaryotic cell on a genome-wide scale.

Spliceosome twin introns in fungal nuclear transcripts.

The spliceosome is an RNA/protein complex, responsible for intron excision from eukaryotic nuclear transcripts. In bacteria, mitochondria and plastids, intron excision does not involve the spliceosome, but occurs through mechanisms dependent on intron RNA secondary and tertiary structure. For group II/III chloroplast introns, "twintrons" (introns within introns) have been described. The excision of the external intron, and thus proper RNA maturation, necessitates prior removal of the internal intron, which interrupts crucial sequences of the former. We have here predicted analogous instances of spliceosomal twintrons ("stwintrons") in filamentous fungi. In two specific cases, where the internal intron interrupts the donor of the external intron after the first or after the second nucleotide, respectively, we show that intermediates with the sequence predicted by the "stwintron" hypothesis, are produced in the splicing process. This implies that two successive rounds of RNA scanning by the spliceosome are necessary to produce the mature mRNA. The phylogenetic distributions of the stwintrons we have identified suggest that they derive from "late" events, subsequent to the appearance of the host intron. They may well not be limited to fungal nuclear transcripts, and their generation and eventual disappearance in the evolutionary process are relevant to hypotheses of intron origin and alternative splicing.

The intracellular galactoglycome in Trichoderma reesei during growth on lactose.

Lactose (1,4-0-ß-D-galactopyranosyl-D-glucose) is used as a soluble carbon source for the production of cellulases and hemicellulases for-among other purposes-use in biofuel and biorefinery industries. The mechanism how lactose induces cellulase formation in T. reesei is enigmatic, however. Previous results from our laboratory raised the hypothesis that intermediates from the two galactose catabolic pathway may give rise to the accumulation of intracellular oligogalactosides that could act as inducer. Here we have therefore used high-performance anion-exchange chromatography-mass spectrometry to study the intracellular galactoglycome of T. reesei during growth on lactose, in T. reesei mutants impaired in galactose catabolism, and in strains with different cellulase productivities. Lactose, allo-lactose, and lactulose were detected in the highest amounts in all strains, and two trisaccharides (Gal-ß-1,6-Gal-ß-1,4-Glc/Fru and Gal-ß-1,4-Gal-ß-1,4-Glc/Fru) also accumulated to significant levels. Glucose and galactose, as well as four further oligosaccharides (Gal-ß-1,3/1,4/1,6-Gal; Gal-ß-1,2-Glc) were only detected in minor amounts. In addition, one unknown disaccharide (Hex-ß-1,1-Hex) and four trisaccharides were also detected. The accumulation of the unknown hexose disaccharide was shown to correlate with cellulase formation in the improved mutant strains as well as the galactose pathway mutants, and Gal-ß-1,4-Gal-ß-1,4-Glc/Fru and two other unknown hexose trisaccharides correlated with cellulase production only in the pathway mutants, suggesting that these compounds could be involved in cellulase induction by lactose. The nature of these oligosaccharides, however, suggests their formation by transglycosylation rather than by glycosyltransferases. Based on our results, the obligate nature of both galactose catabolic pathways for this induction must have another biochemical basis than providing substrates for inducer formation.

Mutations in the Second Alternative Oxidase Gene: A New Approach to Group Aspergillus niger Strains.

Alternative oxidase is a terminal oxidase in the branched mitochondrial electron transport chain of most fungi including Aspergillus niger (subgenus Circumdati, section Nigri). A second, paralogous aox gene (aoxB) is extant in some A. niger isolates but also present in two divergent species of the subgenus Nidulantes-A. calidoustus and A. implicatus-as well as in Penicillium swiecickii. Black aspergilli are cosmopolitan opportunistic fungi that can cause diverse mycoses and acute aspergillosis in immunocompromised individuals. Amongst the approximately 75 genome-sequenced A. niger strains, aoxB features considerable sequence variation. Five mutations were identified that rationally affect transcription or function or terminally modify the gene product. One mutant allele that occurs in CBS 513.88 and A. niger neotype strain CBS 554.65 involves a chromosomal deletion that removes exon 1 and intron 1 from aoxB. Another aoxB allele results from retrotransposon integration. Three other alleles result from point mutations: a missense mutation of the start codon, a frameshift, and a nonsense mutation. A. niger strain ATCC 1015 has a full-length aoxB gene. The A. niger sensu stricto complex can thus be subdivided into six taxa according to extant aoxB allele, which may facilitate rapid and accurate identification of individual species.

Generation, Transfer, and Loss of Alternative Oxidase Paralogues in the Aspergillaceae Family.

Alternative oxidase (Aox) is a terminal oxidase operating in branched electron transport. The activity correlates positively with overflow metabolisms in certain Aspergilli, converting intracellular glucose by the shortest possible path into organic acids, like citrate or itaconate. Aox is nearly ubiquitous in fungi, but aox gene multiplicity is rare. Nevertheless, within the family of the Aspergillaceae and among its various species of industrial relevance-Aspergillus niger, A. oryzae, A. terreus, Penicillium rubens-paralogous aox genes coexist. Paralogous genes generally arise from duplication and are inherited vertically. Here, we provide evidence of four independent duplication events along the lineage that resulted in aox paralogues (aoxB) in contemporary Aspergillus and Penicillium taxa. In some species, three aox genes are co-expressed. The origin of the A. niger paralogue is different than that of the A. terreus paralogue, but all paralogous clades ultimately arise from ubiquitous aoxA parent genes. We found different patterns of uncorrelated gene losses reflected in the Aspergillus pedigree, albeit the original aoxA orthologues persist everywhere and are never replaced. The loss of acquired paralogues co-determines the contemporary aox gene content of individual species. In Aspergillus calidoustus, the two more ancient paralogues have, in effect, been replaced by two aoxB genes of distinct origins.

Manganese(II) ions suppress the transcription of the citrate exporter encoding gene cexA in Aspergillus niger.

Aspergillus niger is an important filamentous fungus used for the industrial production of citric acid. One of the most important factors that affect citric acid production is the concentration of manganese(II) ions present in the culture broth. Under manganese(II)-limiting conditions, the fungus develops a pellet-like morphology that is crucial for high citric acid accumulation. The impact of manganese(II) ions on the transcription of the major citrate exporter encoding gene cexA was studied under manganese(II)-deficient and -sufficient conditions. Furthermore, citric acid production was analyzed in overexpression mutant strains of cexA in the presence and absence of manganese(II) ions, and the influence of CexA on fungal morphology was investigated by microscopy. Transcriptional upregulation of cexA in the absence of manganese(II) ions was observed and, by decoupling cexA expression from the native promoter system, it was possible to secrete more citric acid even in the presence of manganese. This effect was shown for both an inducible and a constitutive overexpression of cexA. Furthermore, it was found that the presence of CexA influences fungal morphology and promotes a more branched phenotype. According to this study, manganese(II) ions suppress transcription of the citrate exporter cexA in Aspergillus niger, causing citric acid secretion to decrease.

Hybrid Vitis Cultivars with American or Asian Ancestries Show Higher Tolerance towards Grapevine Trunk Diseases.

Grape production worldwide is increasingly threatened by grapevine trunk diseases (GTDs). No grapevine cultivar is known to be entirely resistant to GTDs, but susceptibility varies greatly. To quantify these differences, four Hungarian grape germplasm collections containing 305 different cultivars were surveyed to determine the ratios of GTDs based on symptom expression and the proportion of plant loss within all GTD symptoms. The cultivars of monophyletic Vitis vinifera L. origin were amongst the most sensitive ones, and their sensitivity was significantly (p < 0.01) higher than that of the interspecific (hybrid) cultivars assessed, which are defined by the presence of Vitis species other than V. vinifera (e.g., V. labrusca L., V. rupestris Scheele, and V. amurensis Rupr.) in their pedigree. We conclude that the ancestral diversity of grapes confers a higher degree of resilience against GTDs.

Identification of a permease gene involved in lactose utilisation in Aspergillus nidulans.

Lactose is intracellularly hydrolysed by Aspergillus nidulans. Classical mutation mapping data and the physical characteristics of the previously purified glycosyl hydrolase facilitated identification of the clustered, divergently transcribed intracellular ß-galactosidase (bgaD) and lactose permease (lacpA) genes. At the transcript level, bgaD and lacpA were coordinately expressed in response to d-galactose, lactose or l-arabinose, while no transcription was detectable in the additional presence of glucose. In contrast, creA loss-of-function mutants derepressed for both genes to a considerable extent (even) under non-inducing or repressing growth conditions. Lactose- and d-galactose induction nevertheless occurred only in the absence of glucose, indicating a regulatory role for CreA-independent repression. Remarkably, bgaD deletion mutants grew normal on lactose. In contrast, lacpA deletants grew at a much slower rate in lactose liquid medium than wild-type while strains that carried more than one copy of lacpA grew faster, showing that transport is the limiting step in lactose catabolism. The effect of lacpA gene deletion on lactose uptake was exacerbated at lower substrate concentrations, evidence for the existence of a second transport system with a lower affinity for this disaccharide in A. nidulans.

The Biocontrol Potential of Endophytic Trichoderma Fungi Isolated from Hungarian Grapevines, Part II, Grapevine Stimulation.

In the first part of this two-piece publication, the isolation, identification and in vitro characterization of ten endophytic Trichoderma isolates were reported. Here we report the ability of two different mixes of some of these isolates (Trichoderma simmonsii, Trichoderma orientale and Trichoderma gamsii as well as of Trichoderma afroharzianum and T. simmonsii) to colonize and stimulate the growth of grapevines. Two commercial vineyards about 400 km away from the site of isolation were used as experimental fields, from which the strains of three Trichoderma species were re-isolated up to four years after rootstock soaking treatment with conidiospores, performed before planting. The treatments decreased the overall percentage of lost plants of about 30%, although a low number of lost plants (about 5%) were observed also in the control plot. For all cultivars and clones, the Trichoderma treatments significantly increased both the bud burst ratio and bud burst vigor index. In addition, the grape must parameters such as the Brix degrees, as well as the extract, the D-glucose and the D-fructose concentrations all appeared to be improved, suggesting a potentially higher ethanol content of the produced wine. We conclude that grapevine-endophytic Trichoderma isolates promote plant growth, which could be a useful feature for sustainable agriculture in general and integrated plant production in particular.

Unique and Repeated Stwintrons (Spliceosomal Twin Introns) in the Hypoxylaceae.

Introns are usually non-coding sequences interrupting open reading frames in pre-mRNAs [D1,2]. Stwintrons are nested spliceosomal introns, where an internal intron splits a second donor sequence into two consecutive splicing reactions leading to mature mRNA. In Hypoxylon sp. CO27-5, 36 highly sequence-similar [D1,2] stwintrons are extant (sister stwintrons). An additional 81 [D1,2] sequence-unrelated stwintrons are described here. Most of them are located at conserved gene positions rooted deep in the Hypoxylaceae. Absence of exonic sequence bias at the exon-stwintron junctions and a very similar phase distribution were noted for both groups. The presence of an underlying sequence symmetry in all 117 stwintrons was striking. This symmetry, more pronounced near the termini of most of the full-length sister stwintrons, may lead to a secondary structure that brings into close proximity the most distal splice sites, the donor of the internal and the acceptor of the external intron. The Hypoxylon stwintrons were overwhelmingly excised by consecutive splicing reactions precisely removing the whole intervening sequence, whereas one excision involving the distal splice sites led to a frameshift. Alternative (mis)splicing took place for both sister and uniquely occurring stwintrons. The extraordinary symmetry of the sister stwintrons thus seems dispensable for the infrequent, direct utilisation of the distal splice sites.