Identification of potential marker genes for <i>Trichoderma harzianum</i> strains with high antagonistic potential against <i>Rhizoctonia solani</i> 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&#8212;acetyl-xylane esterase AXE1 and endoglucanase Cel61b&#8212;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

Occurrence and Etiology of Brown Apical Necrosis on Persian (English) Walnut Fruit.

In 1998, a severe fruit drop was observed in Italy, principally on cv. Lara Persian (English) walnut (Juglans regia). Dropped fruit showed a brown patch at the blossom end and blackening and rot of inner tissues. The disease, called brown apical necrosis (BAN), was investigated on fruit collected in Italy and France in 1999. In 2000, studies were carried out in three walnut orchards located in Italy and in France to substantiate the etiology of BAN. Isolations performed from inner diseased fruit tissues yielded several fungi, in decreasing frequency of isolation: species of Fusarium and Alternaria, and one species each of Cladosporium, Colletotrichum, and Phomopsis. However, only Fusarium spp. were recovered from stigmas of BAN-affected fruit. The fungi associated with BAN-diseased fruit and species composition differed among locations and over time, confirming results obtained in previous investigations. The species of Fusarium used in pathogenicity tests reproduced BAN-disease symptoms when inoculated on fruit, whereas an Alternaria alternata isolate caused only limited necrosis of the style. However, the role of the other fungi commonly isolated from BAN-diseased fruit remains to be defined. The walnut blight pathogen, Xanthomonas arboricola pv. juglandis, occasionally was isolated from BAN-diseased fruit. No correlation was found between the extent of external brown patches and the size of inner lesions. Repeated isolations from and inoculations of fruit demonstrated that BAN can be considered a complex disease, and the inner infections originate from the style of the fruit

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

Microbial communities and malt quality of durum wheat used in brewing

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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 graminearurn, 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. grarninearum, 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.Ministere de l'Agriculture, de la Viticulture et de la Protection des Consommateurs-Administration des Services Techniques de l'Agriculture; M.I.U.R. Project AGROGEN (Laboratory of GENomics for traits of AGROnomic importance in durum wheat: Identification of useful genes, functional analysis and assisted selection by biological markers for the development of the national seed chain) [602/Ric]; Felix Thornley Cobbold Trust; John Oldacre Foundation; Ministry of Agriculture of the Czech RepublicMinistry of Agriculture, Czech Republic [800415]; Spanish Ministry MINECOSpanish Government [AGL201.4-53928-C2-2-R]; Ministry of Agriculture and Food, Norway; Federal Ministry of Education and Research (BMBF) (GABI-KANADA), BonnFederal Ministry of Education & Research (BMBF) [FKZ 0313711A]; German Academic Exchange Service (DAAD), BonnDeutscher Akademischer Austausch Dienst (DAAD) [A/06/92183]; Finnish Ministry of Agriculture and Forestry; Direction Generale de l'Agriculture, Direction de la Recherche [D31-3159, D31-1162, D31-7055]; P.O.R. SARDEGNA F.S.; Danish Directorate for Food, Fisheries and Agri Business [FFS05-3]; Academy of FinlandAcademy of Finland [126917, 131957, 250904, 252162, 267188, 266984]; Olvi Foundation; Turku University Foundation; CIMO travel grant; Nordic network project New Emerging Mycotoxins and Secondary Metabolites in Toxigenic Fungi of Northern Europe - Nordic Research Board [090014]The Luxembourg institute of Science and Technology, LU, acknowledges the Ministere de l'Agriculture, de la Viticulture et de la Protection des Consommateurs-Administration des Services Techniques de l'Agriculture for financially supporting the Sentinelle project. The work on Italian strains has been financially supported through the M.I.U.R. Project AGROGEN (Laboratory of GENomics for traits of AGROnomic importance in durum wheat: Identification of useful genes, functional analysis and assisted selection by biological markers for the development of the national seed chain) (D. D. 14.03.2005 n. 602/Ric). Funding for the research of Ryan Basler was provided by Felix Thornley Cobbold Trust and the John Oldacre Foundation.; The work of JC was supported by the Ministry of Agriculture of the Czech Republic, Project No. 800415. The research of MG and PG was supported by the Spanish Ministry MINECO (AGL201.4-53928-C2-2-R). The Ministry of Agriculture and Food, Norway funded the work of IH. The research of TM was funded by the Federal Ministry of Education and Research (BMBF) (GABI-KANADA #FKZ 0313711A), Bonn and by the German Academic Exchange Service (DAAD), Bonn (code no.: A/06/92183). PP acknowledges the Finnish Ministry of Agriculture and Forestry for funding the project FinMyco on Fusarium and mycotoxins in Finland. The research of JS was funded by the Direction Generale de l'Agriculture, Direction de la Recherche (ref. D31-3159, D31-1162, D31-7055), in the framework of a project entitled Caracterization et dynamique des fusarioses sur mais en Region Wallonne. BS acknowledges support by P.O.R. SARDEGNA F.S.E. 2007-2013-Obiettivo competitivita regionale e occupazione, Asse IV Capitale umano, Linea di Attivita 1.3.1 (research project Identification of natural and natural-like molecules inhibiting mycotoxin biosynthesis by Fusaria pathogenic on cereals). UT thanks the Danish Directorate for Food, Fisheries and Agri Business grant FFS05-3 for financial support. The work of TY was financially supported by the Academy of Finland (no. 126917, 131957, 250904, 252162, 267188, and 266984), Olvi Foundation, Turku University Foundation, a CIMO travel grant to Taha Hussien, and the Nordic network project New Emerging Mycotoxins and Secondary Metabolites in Toxigenic Fungi of Northern Europe (project 090014), which was funded by the Nordic Research Board

Valutazione della efficacia di un vettore di silenziamento genico per modulare l'espressione dei geni <i>TRI6</i> e <i>TRI5</i> coinvolti nella biosintesi del deossinivalenolo nel fungo fitopatogeno fusarium culmorum

Lo scopo di questo studio è stato quello di analizzare l’espressione differenziale di geni preposti alla biosintesi dei tricoteceni (TRI5 e TRI6) e valutare la produzione di micotossina (DON e il suo derivato acetilato 3Acetil DON) in un ceppo selvaggio di Fusarium culmorum e in ceppi mutanti generati per inserzione di un vettore di silenziamento genico

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

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 (Trich OKEY), 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

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.Fil: Oufensou, Safa. Università Degli Studi Di Sassari; ItaliaFil: Scherm, Barbara. Università Degli Studi Di Sassari; ItaliaFil: Pani, Giovanna. Università Degli Studi Di Sassari; ItaliaFil: Balmas, Virgilio. Università Degli Studi Di Sassari; ItaliaFil: Fabbri, Davide. Istituto Di Chimica Biomolecolare; ItaliaFil: Dettori, Maria Antonietta. Istituto Di Chimica Biomolecolare; ItaliaFil: Carta, Paola. Istituto Di Chimica Biomolecolare; ItaliaFil: Malbrán, Ismael. Universidad Nacional de La Plata. Facultad de Ciencias Agrarias y Forestales. Departamento de Ciencias Biológicas. Centro de Investigaciones de Fitopatología. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigaciones de Fitopatología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Migheli, Quirico. Università Degli Studi Di Sassari; ItaliaFil: Delogu, Giovanna. Istituto Di Chimica Biomolecolare; Itali

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