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

Analyse Hitzeschockprotein-kodierender Gene von Mykoplasmen

[no abstract

Biodiversity of the genus Trichoderma and identification of marker genes involved in the antagonism between Trichoderma spp. and plant pathogenic fungi

As Mediterranean islands are known for their high diversity of vascular plants and a high level of endemism, they can provide basic insights into the relationship between geographical patterns and ecological processes. Studying the soil fungus Trichoderma/Hypocrea is of special interest to our working group due to its world-wide distribution, the continuos ongoing identification of new isolates, and its high percentage of strains being effective as biocontrol agents. We isolated 482 strains of Hypocrea/Trichoderma from 15 soils comprising undisturbed and disturbed soil environments. Isolates were identified at the species level by the oligonucleotide BarCode for Hypocrea/Trichoderma, sequence similarity analysis 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. T. harzianum (H. lixii) is well known as a biocontrol agent against a range of economically important plant pathogens. In the present study we used a Rapid Subtraction Hybridisation approach to isolate genes which are differentially expressed during mycelial contact between T. harzianum and R. solani, and could serve as marker genes for selection of superior biocontrol strains. The 50 characterised clones comprised proteins involved (i) in protein synthesis and turn-over, (ii) in metabolism, and (iii) in the hydrolysis of macromolecules. An acetyl xylane esterase and an endoglucanase showed significant upregulation during in vivo confrontation of a T. harzianum strain with high antagonistic capability towards R. solani, while the expression was low or absent in a T. harzianum strain with poor antagonistic activity

Genetic Diversity of Fusarium oxysporum f. sp. dianthi in Southern Spain

The diversity of races and prevalence of pathogenic populations of Fusarium oxysporum f. sp. dianthi (Fod) were surveyed in an area in southern Spain. From 54 farms, 132 isolates were collected from wilted carnation plants. Isolates were characterized by RAPD-PCR, DNA sequence analysis of the TEF1-α gene, and race-specific molecular markers. Selected isolates from RAPD groups were phenotypically evaluated by pathogenicity tests. Data analysis showed that Fod race 2 was the most frequent and prevalent race in the study area, followed by race 1/8. Moreover, phylogenetic analyses showed similar results, which were different to those of the race-specific PCR assays. It was concluded that (i) seven isolates were not classified in groups where Fod testers were clustered; even they showed different results when race-specific markers were used, (ii) ten isolates with retarded race 1 or race 8 specific band were characterized as F. proliferatum by TEF1-α gene sequencing and clustered into an outgroup, and (iii) six isolates failed to generate an amplification signal using race-specific markers. Furthermore, three of them were grouped close to race 2 tester according to the phylogenetic analyses, showing the same differential pathogenicity as race 2. This may indicate a Fod race 2 subgroup in this region.Ministerio de Educación y Ciencia AGL2005-08137-C03-02Junta de Andalucía P06-AGR-02313Ministerio de Ciencia e Innovación AGL2008-05414-C03-0

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

Natural phenolic inhibitors of trichothecene biosynthesis by the wheat fungal pathogen <i>Fusarium culmorum</i>: 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

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