Water distribution systems in Sardinian hospitals host invasive clonal lineages of the Fusarium oxysporum and Fusarium solani species complexes.

Several Fusarium species cause disease on human hosts, including commonly fatal infections in immunocompromised individuals. Recently, cases of hospitalized patients affected by fusaria were reported in the Tyrrhenian Island of Sardinia, Italy. To precisely characterize the Fusarium species and haplotypes present in hospitals of the region, a multilocus DNA sequence typing (MLST) approach was applied. Water distribution systems in four departments belonging to four Sardinian hospitals were sampled. Fusarium species and sequence types (STs) were identified using MLST based on sequences of the elongation factor 1-alpha (EF-1α) gene, the nuclear ribosomal DNA intergenic spacer region (IGS rDNA), and/or a portion of the second-largest subunit of RNA polymerase (RPB2) gene. The majority of isolates obtained from Sardinian hospitals (90.7%) were identified as representatives of the Fusarium oxysporum species complex (FOSC), followed by those of the F. solani species complex (FSSC) (8.2%), and F. dimerum (1.1% of all isolates). Ten STs were found among the FOSC and FSSC, with more than 60% of the isolates identified as either FOSC ST 33 or FSSC 1 (F. petroliphilum). More than half of the FOSC isolates obtained from the water systems in all four hospitals belonged to the worldwide distributed clonal lineage ST 33. This haplotype is the most prevalent among the FOSC in different countries, being responsible for the vast majority of cases of human fusariosis.

Clonality, spatial structure, and pathogenic variation in Fusarium fujikuroi from rain-fed rice in southern Laos.

Bakanae disease, caused by the fungal phytopathogen Fusarium fujikuroi, can be detected in most rice (Oryza sativa L.) growing areas worldwide. In this study, we investigated the population structure of this fungus in southern Lao PDR, a country located near the geographic origin of rice domestication. Microsatellites (SSRs) and mating type (MAT) analyses, pathogenicity and fungicide sensitivity tests were integrated in the study. The first key finding is that the population genetic structure of F. fujikuroi in Lao PDR is consistent with high clonal reproduction. Indeed, (i) "true" clones were identified; (ii) within populations, MAT types were frequently skewed from 1:1 ratio, (iii) linkage disequilibrium (among SSRs as also among SSRs and MAT) was present, and (iv) gene-flow between opposite MAT types within the same population is restricted. The presence of genetic divergence among areas and populations and the occurrence of positive spatial autocorrelation of genetic variation, indicate that migration is restricted, and that genetic drift plays an important role in the evolution of this fungus. Two main well-defined groups of isolates were detected (FST = 0.213) that display a non-random spatial distribution. They differ in the ability to induce seedlings death but not seedlings elongation (the typical Bakanae symptom) suggesting that the pathogen's ability to induce the two symptoms is under different genetic control. Finally, we compared two agroecosystems with contrasting characteristics: low-input and traditional (Lao PDR) vs high-input and modern (Italy). We found differences in the level of population structuring and of spatial autocorrelation. This suggests that the evolutionary potential of the fungus not only depends on its intrinsic characteristics, but is strongly influenced by other external factors, most likely by the dynamics of infested seed exchange. Thus, quarantine and chemical treatments are a way to reduce population connectivity and hence the evolutionary potential of this pathogen.

Honokiol, magnolol and its monoacetyl derivative show strong anti-fungal effect on Fusarium isolates of clinical relevance.

The antifungal activity of magnolol and honokiol, two naturally occurring hydroxylated biphenyls, and of their synthetic derivatives was evaluated on a collection of representative isolates of Fusarium oxysporum, F. solani and F. verticillioides of clinical and ecological concern. The tested compounds were proposed as a 'natural' alternative to conventional fungicides, even though a larger range of concentrations (5-400 µg/ml) was applied. The activity of magnolol and honokiol was compared with that of terbinafine (0.1-10 µg/ml), and fluconazole (1-50 µg/ml), two fungicides widely used in treating fungal infections on humans. Magnolol showed similar fungicidal activity compared to fluconazole, whereas honokiol was more effective in inhibiting mycelium growth compared to this fungicide on all tested clinical Fusarium spp. isolates. Compared to terbinafine, honokiol showed similar antifungal activity when tested on clinical F. solani isolates, whereas magnolol was less effective at all selected concentrations (5-400 µg/ml). The different position of the phenol-OH group, as well as its protection, explain different in vitro activities between magnolol, honokiol, and their derivatives. Furthermore, magnolol showed mycelium dry weight reduction at a concentration of 0.5 mM when tested on a set of agricultural isolates of Fusaria, leading to complete inhibition of some of them. Magnolol and honokiol are proposed as efficient and safe candidates for treating clinically relevant Fusaria.

FcRav2, a gene with a ROGDI domain involved in Fusarium head blight and crown rot on durum wheat caused by Fusarium culmorum.

Fusarium culmorum is a soil-borne fungal pathogen which causes foot and root rot and Fusarium head blight on small-grain cereals, in particular wheat and barley. It causes significant yield and quality losses and results in the contamination of kernels with type B trichothecene mycotoxins. Our knowledge of the pathogenicity factors of this fungus is still limited. A transposon tagging approach based on the mimp1/impala double-component system has allowed us to select a mutant altered in multiple metabolic and morphological processes, trichothecene production and virulence. The flanking regions of mimp1 were used to seek homologies in the F. culmorum genome, and revealed that mimp1 had reinserted within the last exon of a gene encoding a hypothetical protein of 318 amino acids which contains a ROGDI-like leucine zipper domain, supposedly playing a protein-protein interaction or regulatory role. By functional complementation and bioinformatic analysis, we characterized the gene as the yeast Rav2 homologue, confirming the high level of divergence in multicellular fungi. Deletion of FcRav2 or its orthologous gene in F. graminearum highlighted its ability to influence a number of functions, including virulence, trichothecene type B biosynthesis, resistance to azoles and resistance to osmotic and oxidative stress. Our results indicate that the FcRav2 protein (and possibly the RAVE complex as a whole) may become a suitable target for new antifungal drug development or the plant-mediated resistance response in filamentous fungi of agricultural interest.

Natural Phenolic Inhibitors of Trichothecene Biosynthesis by the Wheat Fungal Pathogen Fusarium culmorum: A Computational Insight into the Structure-Activity Relationship.

A model of the trichodiene synthase (TRI5) of the wheat fungal pathogen and type-B trichothecene producer Fusarium culmorum was developed based on homology modelling with the crystallized protein of F. sporotrichioides. Eight phenolic molecules, namely ferulic acid 1, apocynin 2, propyl gallate 3, eugenol 4, Me-dehydrozingerone 5, eugenol dimer 6, magnolol 7, and ellagic acid 8, were selected for their ability to inhibit trichothecene production and/or fungal vegetative growth in F. culmorum. The chemical structures of phenols were constructed and partially optimised based on Molecular Mechanics (MM) studies and energy minimisation by Density Functional Theory (DFT). Docking analysis of the phenolic molecules was run on the 3D model of F. culmorum TRI5. Experimental biological activity, molecular descriptors and interacting-structures obtained from computational analysis were compared. Besides the catalytic domain, three privileged sites in the interaction with the inhibitory molecules were identified on the protein surface. The TRI5-ligand interactions highlighted in this study represent a powerful tool to the identification of new Fusarium-targeted molecules with potential as trichothecene inhibitors.

A European Database of Fusarium graminearum and F. culmorum Trichothecene Genotypes.

Fusarium species, particularly Fusarium graminearum and F. culmorum, are the main cause of trichothecene type B contamination in cereals. Data on the distribution of Fusarium trichothecene genotypes in cereals in Europe are scattered in time and space. Furthermore, a common core set of related variables (sampling method, host cultivar, previous crop, etc.) that would allow more effective analysis of factors influencing the spatial and temporal population distribution, is lacking. Consequently, based on the available data, it is difficult to identify factors influencing chemotype distribution and spread at the European level. Here we describe the results of a collaborative integrated work which aims (1) to characterize the trichothecene genotypes of strains from three Fusarium species, collected over the period 2000-2013 and (2) to enhance the standardization of epidemiological data collection. Information on host plant, country of origin, sampling location, year of sampling and previous crop of 1147 F. graminearum, 479 F. culmorum, and 3 F. cortaderiae strains obtained from 17 European countries was compiled and a map of trichothecene type B genotype distribution was plotted for each species. All information on the strains was collected in a freely accessible and updatable database (www.catalogueeu.luxmcc.lu), which will serve as a starting point for epidemiological analysis of potential spatial and temporal trichothecene genotype shifts in Europe. The analysis of the currently available European dataset showed that in F. graminearum, the predominant genotype was 15-acetyldeoxynivalenol (15-ADON) (82.9%), followed by 3-acetyldeoxynivalenol (3-ADON) (13.6%), and nivalenol (NIV) (3.5%). In F. culmorum, the prevalent genotype was 3-ADON (59.9%), while the NIV genotype accounted for the remaining 40.1%. Both, geographical and temporal patterns of trichothecene genotypes distribution were identified.

Natural and natural-like phenolic inhibitors of type B trichothecene in vitro production by the wheat (Triticum sp.) pathogen Fusarium culmorum.

Fusarium culmorum, a fungal pathogen of small grain cereals, produces 4-deoxynivalenol and its acetylated derivatives that may cause toxicoses on humans or animals consuming contaminated food or feed. Natural and natural-like compounds belonging to phenol and hydroxylated biphenyl structural classes were tested in vitro to determine their activity on vegetative growth and trichothecene biosynthesis by F. culmorum. Most of the compounds tested at 1.5 or 1.0 mM reduced 3-acetyl-4-deoxynivalenol production by over 70% compared to the control, without affecting fungal growth significantly. Furthermore, several compounds retained their ability to inhibit toxin in vitro production at the lowest concentrations of 0.5 and 0.25 mM. Magnolol 27 showed fungicidal activity even at 0.1 mM. No linear correlation was observed between antioxidant properties of the compounds and their ability to inhibit fungal growth and mycotoxigenic capacity. A guaiacyl unit in the structure may play a key role in trichothecene inhibition.

FcStuA from Fusarium culmorum controls wheat foot and root rot in a toxin dispensable manner.

Fusarium culmorum is one of the most harmful pathogens of durum wheat and is the causal agent of foot and root rot (FRR) disease. F. culmorum produces the mycotoxin deoxynivalenol (DON) that is involved in the pathogenic process. The role of the gene FcStuA, a StuA ortholog protein with an APSES domain sharing 98.5% homology to the FgStuA protein (FGSG10129), was determined by functional characterisation of deletion mutants obtained from two F. culmorum wild-type strains, FcUk99 (a highly pathogenic DON producer) and Fc233B (unable to produce toxin and with a mild pathogenic behavior). The ΔFcStuA mutants originating from both strains showed common phenotypic characters including stunted vegetative growth, loss of hydrophobicity of the mycelium, altered pigmentation, decreased activity of polygalacturonic enzymes and catalases, altered and reduced conidiation, delayed conidial germination patterns and complete loss of pathogenicity towards wheat stem base/root tissue. Glycolytic process efficiency [measured as growth on glucose as sole carbon (C) source] was strongly impaired and growth was partially restored on glutamic acid. Growth on pectin-like sources ranked in between glucose and glutamic acid with the following order (the lowest to the highest growth): beechwood xylan, sugarbeet arabinan, polygalacturonic acid, citrus pectin, apple pectin, potato azogalactan. DON production in the mutants originating from FcUK99 strain was significantly decreased (-95%) in vitro. Moreover, both sets of mutants were unable to colonise non-cereal plant tissues, i.e. apple and tomato fruits and potato tubers. No differences between mutants, ectopic and wild-type strains were observed concerning the level of resistance towards four fungicides belonging to three classes, the demethylase inhibitors epoxiconazole and tebuconzole, the succinate dehydrogenase inhibitor isopyrazam and the cytochrome bc1 inhibitor trifloxystrobin. StuA, given its multiple functions in cell regulation and pathogenicity control, is proposed as a potential target for novel disease management strategies.

Fusarium culmorum: causal agent of foot and root rot and head blight on wheat.

UNLABELLED: Fusarium culmorum is a ubiquitous soil-borne fungus able to cause foot and root rot and Fusarium head blight on different small-grain cereals, in particular wheat and barley. It causes significant yield and quality losses and results in contamination of the grain with mycotoxins. This review summarizes recent research activities related to F. culmorum, including studies into its population diversity, mycotoxin biosynthesis, mechanisms of pathogenesis and resistance, the development of diagnostic tools and preliminary genome sequence surveys. We also propose potential research areas that may expand our basic understanding of the wheat-F. culmorum interaction and assist in the management of the disease caused by this pathogen. TAXONOMY: Fusarium culmorum (W.G. Smith) Sacc. Kingdom Fungi; Phylum Ascomycota; Subphylum Pezizomycotina; Class Sordariomycetes; Subclass Hypocreomycetidae; Order Hypocreales; Family Nectriaceae; Genus Fusarium. DISEASE SYMPTOMS: Foot and root rot (also known as Fusarium crown rot): seedling blight with death of the plant before or after emergence; brown discoloration on roots and coleoptiles of the infected seedlings; brown discoloration on subcrown internodes and on the first two/three internodes of the main stem; tiller abortion; formation of whiteheads with shrivelled white grains; Fusarium head blight: prematurely bleached spikelets or blighting of the entire head, which remains empty or contains shrunken dark kernels. IDENTIFICATION AND DETECTION: Morphological identification is based on the shape of the macroconidia formed on sporodochia on carnation leaf agar. The conidiophores are branched monophialides, short and wide. The macroconidia are relatively short and stout with an apical cell blunt or slightly papillate; the basal cell is foot-shaped or just notched. Macroconidia are thick-walled and curved, usually 3-5 septate, and mostly measuring 30-50 × 5.0-7.5 µm. Microconidia are absent. Oval to globose chlamydospores are formed, intercalary in the hyphae, solitary, in chains or in clumps; they are also formed from macroconidia. The colony grows very rapidly (1.6-2.2 cm/day) on potato dextrose agar (PDA) at the optimum temperature of 25 °C. The mycelium on PDA is floccose, whitish, light yellow or red. The pigment on the reverse plate on PDA varies from greyish-rose, carmine red or burgundy. A wide array of polymerase chain reaction (PCR) and real-time PCR tools, as well as complementary methods, which are summarised in the first two tables, have been developed for the detection and/or quantification of F. culmorum in culture and in naturally infected plant tissue. HOST RANGE: Fusarium culmorum has a wide range of host plants, mainly cereals, such as wheat, barley, oats, rye, corn, sorghum and various grasses. In addition, it has been isolated from sugar beet, flax, carnation, bean, pea, asparagus, red clover, hop, leeks, Norway spruce, strawberry and potato tuber. Fusarium culmorum has also been associated with dermatitis on marram grass planters in the Netherlands, although its role as a causal agent of skin lesions appears questionable. It is also isolated as a symbiont able to confer resistance to abiotic stress, and has been proposed as a potential biocontrol agent to control the aquatic weed Hydrilla spp. USEFUL WEBSITES: http://isolate.fusariumdb.org/; http://sppadbase.ipp.cnr.it/; http://www.broad.mit.edu/annotation/genome/fusarium_group/MultiHome.html; http://www.fgsc.net/Fusarium/fushome.htm; http://plantpath.psu.edu/facilities/fusarium-research-center; http://www.phi-base.org/; http://www.uniprot.org/; http://www.cabi.org/; http://www.indexfungorum.org/

Transposition of the miniature inverted-repeat transposable element mimp1 in the wheat pathogen Fusarium culmorum.

High-throughput methods are needed for functional genomics analysis in Fusarium culmorum, the cause of crown and foot rot on wheat and a type B trichothecene producer. Our aim was to develop and test the efficacy of a double-component system based on the ability of the impala transposase to transactivate the miniature inverted-repeat transposable element mimp1 of Fusarium oxysporum. We report, for the first time, the application of a tagging system based on a heterologous transposon and of splinkerette-polymerase chain reaction to identify mimp1 flanking regions in the filamentous fungus F. culmorum. Similar to previous observations in Fusarium graminearum, mimp1 transposes in F. culmorum by a cut-and-paste mechanism into TA dinucleotides, which are duplicated on insertion. mimp1 was reinserted in open reading frames in 16.4% (i.e. 10 of 61) of the strains analysed, probably spanning throughout the entire genome of F. culmorum. The effectiveness of the mimp1/impala double-component system for gene tagging in F. culmorum was confirmed phenotypically for a putative aurofusarin gene. This system also allowed the identification of two genes putatively involved in oxidative stress-coping capabilities in F. culmorum, as well as a sequence specific to this fungus, thus suggesting the valuable exploratory role of this tool.

Identification of differentially expressed genes associated with changes in the morphology of Pichia fermentans on apple and peach fruit.

Pichia fermentans (strain DISAABA 726) is an effective biocontrol agent against Monilinia fructicola and Botrytis cinerea when inoculated in artificially wounded apple fruit but is an aggressive pathogen when inoculated on wounded peach fruit, causing severe fruit decay. Pichia fermentans grows as budding yeast on apple tissue and exhibits pseudohyphal growth on peach tissue, suggesting that dimorphism may be associated with pathogenicity. Two complementary suppressive subtractive hybridization (SSH) strategies, that is, rapid subtraction hybridization (RaSH) and PCR-based subtraction, were performed to identify genes differentially expressed by P. fermentans after 24-h growth on apple vs. peach fruit. Gene products that were more highly expressed on peach than on apple tissue, or vice versa, were sequenced and compared with available yeast genome sequence databases. Several of the genes more highly expressed, when P. fermentans was grown on peach, were related to stress response, glycolysis, amino acid metabolism, and alcoholic fermentation but surprisingly not to cell wall degrading enzymes such as pectinases or cellulases. The dual activity of P. fermentans as both a biocontrol agent and a pathogen emphasizes the need for a thorough risk analysis of potential antagonists to avoid unpredictable results that could negatively impact the safe use of postharvest biocontrol strategies.

Altered trichothecene biosynthesis in TRI6-silenced transformants of Fusarium culmorum influences the severity of crown and foot rot on durum wheat seedlings.

An RNA silencing construct was used to alter mycotoxin production in the plant pathogenic fungus Fusarium culmorum, the incitant of crown and foot rot on wheat. The transformation of a wild-type strain and its nitrate reductase-deficient mutant with inverted repeat transgenes (IRTs) containing sequences corresponding to the trichothecene regulatory gene TRI6 was achieved using hygromycin B resistance as a selectable marker. Southern analysis revealed a variety of integration patterns of the TRI6 IRT. One transformant underwent homologous recombination with deletion of the endogenous TRI6 gene, whereas, in another transformant, the TRI6 IRT was not integrated into the genome. The TRI6 IRT did not alter the physiological characteristics, such as spore production, pigmentation or growth rate, on solid media. In most transformants, a high TRI6 amplification signal was detected by quantitative reverse transcription-polymerase chain reaction, corresponding to a TRI6-hybridizing smear of degraded fragments by Northern analysis, whereas TRI5 expression decreased compared with the respective nontransformed strain. Four transformants showed increased TRI5 expression, which was correlated with a dramatic (up to 28-fold) augmentation of deoxynivalenol production. Pathogenicity assays on durum wheat seedlings confirmed that impairment of deoxynivalenol production in the TRI6 IRT transformants correlated with a loss of virulence, with decreased disease indices ranging from 40% to 80% in nine silenced strains, whereas the overproducing transformants displayed higher virulence compared with the wild-type.

Multilocus phylogenetics show high levels of endemic fusaria inhabiting Sardinian soils (Tyrrhenian Islands).

The Mediterranean island of Sardinia is well known for high levels of vascular plant diversity and endemism, but little is known about its microbial diversity. Under the hypothesis that Fusarium species would show similarly high diversity, we estimated variability in Fusarium species composition among 10 sites around the island. Markers previously adopted for multilocus sequence typing (MLST) were used to determine multilocus DNA sequence haplotypes for 263 Fusarium isolates. In addition portions of the translation elongation factor 1-alpha and second largest RNA polymerase subunit genes were sequenced for all isolates. The intergenic spacer (IGS) region of the nuclear ribosomal RNA gene repeat was sequenced for members of the F. oxysporum species complex (FOSC), and a portion of the nuclear ribosomal RNA gene repeat comprising the internal transcribed spacer (ITS) and part of the large nuclear ribosomal RNA subunit was sequenced for members of the F. solani species complex (FSSC). Seventy-three multilocus haplotypes were identified among the 263 isolates typed, of which 48 represented FOSC and FSSC. Thirty-seven of 48 FOSC two-locus and FSSC three-locus haplotypes had not been observed previously. The 38 non-FOSC/FSSC fusaria comprised 25 haplotypes distributed among 10 species, five of which appear to represent novel, phylogenetically distinct species. In general newly discovered haplotypes were restricted to one or a few sites. All FSSC isolates represented new haplotypes in phylogenetic species FSSC 5 and 9, which differ from the phylogenetic species dominant in soils worldwide. No obvious correlations were found between haplotype diversity and geospatial or habitat distribution. Overall these results indicate a high degree of Fusarium genetic diversity on multiple geographic scales within Sardinia. These results contrast with recent work showing that common, cosmopolitan species dominate Sardinia's Trichoderma biodiversity. All data are available for access and viewing from the FUSARIUM-ID database.

Molecular phylogenetic diversity of dermatologic and other human pathogenic fusarial isolates from hospitals in northern and central Italy.

Fifty-eight fusaria isolated from 50 Italian patients between 2004 and 2007 were subject to multilocus DNA sequence typing to characterize the spectrum of species and circulating sequence types (STs) associated with dermatological infections, especially onychomycoses and paronychia, and other fusarioses in northern and central Italy. Sequence typing revealed that the isolates were nearly evenly divided among the Fusarium solani species complex (FSSC; n = 18), the F. oxysporum species complex (FOSC; n = 20), and the Gibberella (Fusarium) fujikuroi species complex (GFSC; n = 20). The three-locus typing scheme used for members of the FSSC identified 18 novel STs distributed among six phylogenetically distinct species, yielding an index of discrimination of 1.0. Phylogenetic analysis of the FOSC two-locus data set identified nine STs, including four which were novel, and nine isolates of ST 33, the previously described widespread clonal lineage. With the inclusion of eight epidemiologically unrelated ST 33 isolates, the FOSC typing scheme scored a discrimination index of 0.787. The two-locus GFSC typing scheme, which was primarily designed to identify species, received the lowest discrimination index, with a score of 0.492. The GFSC scheme, however, was used to successfully identify 17 isolates as F. verticillioides, 2 as F. sacchari, and 1 as F. guttiforme. This is the first report that F. guttiforme causes a human mycotic infection, which was supported by detailed morphological analysis. In addition, the results of a pathogenicity experiment revealed that the human isolate of F. guttiforme was able to induce fusariosis of pineapple, heretofore its only known host.

Identification of potential marker genes for Trichoderma harzianum strains with high antagonistic potential against Rhizoctonia solani by a rapid subtraction hybridization approach.

A rapid subtraction hybridization approach was used to isolate genes differentially expressed during mycelial contact between Trichoderma harzianum (Hypocrea lixii) and Rhizoctonia solani, and could serve as marker genes for selection of superior biocontrol strains. Putatively positive clones were evaluated by transcription analysis during mycelial contact with R. solani versus growth on glucose, and for their differential transcription between two strains with either strong or poor biocontrol capability before, at, and after contact with R. solani. Besides four clones, which had similarity to putative but as yet uncharacterized proteins, they comprised ribosomal proteins, proteins involved in transcriptional switch and regulation, amino acid and energy catabolism, multidrug resistance, and degradation of proteins and glucans. Transcription of three clones was evaluated in five T. harzianum strains under confrontation conditions with R. solani. Two clones-acetyl-xylane esterase AXE1 and endoglucanase Cel61b-showed significant upregulation during in vivo confrontation of a T. harzianum strain that successively demonstrated a very high antagonistic capability towards R. solani, while expression was progressively lower in a series of T. harzianum strains with intermediate to poor antagonistic activity. These clones are promising candidates for use as markers in the screening of improved T. harzianum biocontrol strains.

Soils of a Mediterranean hot spot of biodiversity and endemism (Sardinia, Tyrrhenian Islands) are inhabited by pan-European, invasive species of Hypocrea/Trichoderma.

We have used a Mediterranean hot spot of biodiversity (the Island of Sardinia) to investigate the impact of abiotic factors on the distribution of species of the common soil fungus Trichoderma. To this end, we isolated 482 strains of Hypocrea/Trichoderma from 15 soils comprising undisturbed and disturbed environments (forest, shrub lands and undisturbed or extensively grazed grass steppes respectively). Isolates were identified at the species level by the oligonucleotide BarCode for Hypocrea/Trichoderma (TrichOKEY), sequence similarity analysis (Trichoblast) and phylogenetic inferences. The majority of the isolates were positively identified as pan-European and/or pan-global Hypocrea/Trichoderma species from sections Trichoderma and Pachybasium, comprising H. lixii/T. harzianum, T. gamsii, T. spirale, T. velutinum, T. hamatum, H. koningii/T. koningii, H. virens/T. virens, T. tomentosum, H. semiorbis, H. viridescens/T. viridescens, H. atroviridis/T. atroviride, T. asperellum, H. koningiopsis/T. koningiopsis and Trichoderma sp. Vd2. Only one isolate represented a new, undescribed species belonging to the Harzianum-Catoptron Clade. Internal transcribed spacer sequence analysis revealed only one potentially endemic internal transcribed spacer 1 allele of T. hamatum. All other species exhibited genotypes that were already found in Eurasia or in other continents. Only few cases of correlation of species occurrence with abiotic factors were recorded. The data suggest a strong reduction of native Hypocrea/Trichoderma diversity, which was replaced by extensive invasion of species from Eurasia, Africa and the Pacific Basin.

The strange case of a biofilm-forming strain of Pichia fermentans, which controls Monilinia brown rot on apple but is pathogenic on peach fruit.

A biofilm-forming strain of Pichia fermentans proved to be most effective in controlling brown rot on apple fruit when coinoculated into artificial wounds with a phytopathogenic isolate of Monilinia fructicola. Culture filtrates and autoclaved cells had no significant influence on the disease. When sprayed onto the apple fruit surface, this yeast formed a thin biofilm but failed to colonize the underlying tissues. When inoculated into wounds artificially inflicted to peach fruit or when sprayed onto the surface of peach fruit, the same strain showed an unexpected pathogenic behaviour, causing rapid decay of fruit tissues even in the absence of M. fructicola. Both optical and scanning electron microscopy were used to evaluate the pattern of fruit tissue colonization by P. fermentans. While on apple surface and within the apple wound the antagonist retained its yeast-like shape, colonization of peach fruit tissue was always characterized by a transition from budding growth to pseudohyphal growth. These results suggest that pseudohyphal growth plays a major role in governing the potential pathogenicity of P. fermentans, further emphasizing the importance of a thorough risk assessment for the safe use of any novel biocontrol agent.

Antagonism of Pythium blight of zucchini by Hypocrea jecorina does not require cellulase gene expression but is improved by carbon catabolite derepression.

Toward a better understanding of the biochemical events that lead to biocontrol of plant pathogenic fungi by Hypocrea/Trichoderma spp., we investigated the importance of carbon catabolite (de)repression and cellulase formation in the antagonization of Pythium ultimum by Hypocrea jecorina (Trichoderma reesei) on agar plates and in planta. Hypocrea jecorina QM9414 could antagonize and overgrow P. ultimum but not Rhizoctonia solani in plate confrontation tests, and provided significant protection of zucchini plants against P. ultimum blight in planta. A carbon catabolite derepressed cre1 mutant of H. jecorina antagonized P. ultimum on plates more actively and increased the survival rates of P. ultimum-inoculated zucchini plants in comparison with strain QM9414. A H. jecorina mutant impaired in cellulase induction could also antagonize P. ultimum on plates and provided the same level of protection of zucchini plants against P. ultimum as strain QM9414 did. We conclude that cellulase formation is dispensable for biocontrol of P. ultimum, whereas carbon catabolite derepression increases the antagonistic ability by apparently acting on other target genes.

Residue level, persistence, and storage performance of citrus fruit treated with fludioxonil.

The potential of postharvest dip treatments with fludioxonil (FLU) (a synthetic analogue of the bacterial metabolite of pyrrolnitrin), in controlling postharvest decay caused by Penicillium digitatum and Penicillium italicum of citrus fruit was investigated in comparison with the conventional fungicide imazalil (IMZ). The ultrastructural changes of fruit epicuticular wax was investigated as a function of water dip temperature, and the possible role of these changes was related to residue accumulation under FLU treatment. Residues retained by fruit were determined as a function of fungicide concentration, dip temperature, and fruit storage conditions. Scanning electron microscopy analysis revealed that fruit dipping in water at 30 or 40 degrees C did not cause differences in cuticular wax's ultrastructure in comparison to control fruit, while treatments at 50, 55, or 60 degrees C caused the disappearance of wax platelets, resulting in relatively homogeneous skin surface, due to partial "melting" of epicuticular wax. Residues of FLU in fruit treated at 20 or 50 degrees C were significantly correlated with the doses of fungicide applied. When equal amounts of fungicide were employed, the residue concentrations were notably higher (from 2.6- to 4-fold) in fruit treated at 50 degrees C than in fruit treated at 20 degrees C. The dissipation rate of FLU in "Salustiana" and "Tarocco" oranges was lower in fruit subjected to treatment at 50 degrees C. The minimal FLU concentration for almost complete decay control in artificially wounded fruit during 7-d storage at 20 degrees C was 400 mg/L active ingredient (ai) in fruit treated at 20 degrees C and 100 mg/L ai in fruit treated at 50 degrees C. Results on nonwounded Tarocco oranges subjected to 3 weeks of simulated quarantine conditions at 1 degrees C, plus 6 weeks of standard storage at 8 degrees C and an additional two weeks of simulated marketing period (SMP) at 20 degrees C revealed that almost complete decay control with FLU applications of 100 mg/L at 50 degrees C and 400 mg/L at 20 degrees C resulted in ca. 0.8 mg/kg FLU fruit residues, in agreement with results on wounded citrus fruit. When equal concentrations and temperatures were applied, FLU treatments were as effective as IMZ. In vitro trials showed a low sensitivity to FLU against P. digitatum and P. italicum isolates. MIC values for the complete inhibition of mycelium growth were >or=100 microg/mL, while ED(50) values ranged from 0.1 to 1 microg/mL for P. digitatum and from 1 to >100 microg/mL for P. italicum. The latter result suggests that care should be taken to avoid exclusive application of FLU in a sustainable program for management of fruit decay. However, integrating fungicide application and hot water dip may reduce the possibility of selecting fungicide-resistant populations of the pathogen, by increasing the effectiveness of the treatment.

Detection of transcripts of the aflatoxin genes aflD, aflO, and aflP by reverse transcription-polymerase chain reaction allows differentiation of aflatoxin-producing and non-producing isolates of Aspergillus flavus and Aspergillus parasiticus.

The aim of this study was to test the suitability of the RT-PCR (reverse transcription-polymerase chain reaction) technique to differentiate aflatoxin-producing from aflatoxin-non-producing strains of Aspergillus flavus and Aspergillus parasiticus. Total RNAs of 13 strains grown under inducing yeast extract-sucrose (YES) and non-inducing yeast extract-peptone (YEP) media, respectively, were analyzed by using specific primers based on the conserved regions of nine structural genes (aflD, aflG, aflH, aflI, aflK, aflM, aflO, aflP, and aflQ) and two regulatory genes aflS and aflR of the aflatoxin B1 biosynthetic pathway. Transcription was confirmed by the expression of the beta-tubulin gene. The expression of the majority aflatoxin biosynthetic genes including aflR and aflS of all strains varied with regard to the aflatoxin-producing ability and the growth conditions. Nonetheless, we found that the expression profile of the three genes aflD, aflO, and aflP was consistently correlated with a strain's ability to produce aflatoxins or not in YES as well as the inability to produce aflatoxins in YEP. The devised RT-PCR profiling method reflects aflatoxin concentrations ranging from 0.1 to 60 microg/ml of the culture filtrates as determined by fluorescence HPLC. The results are discussed in relation to the suitability of RT-PCR as well as cDNA-based array techniques in diagnostic laboratory settings where individual isolates are being tested for potential toxin production to identify toxigenic isolates of Aspergillus species.