SPPADBASE: the first on-line searchable database of PCR primers for phytopathogenic fungi

The fast and unambiguous identification of microbial pathogens affecting plants or plant products is an essential prerequisite for obtaining high-quality and safe production. Ecologically friendly practice of the modern agriculture requires the adoption of diagnostic techniques able to detect minimum inoculum levels of pathogens in soil, seeds, transplants or crops, to limit the raise of epidemics and to address the adoption of rational and efficient control means. Moreover, there is an increasing public and official awareness of the potential threat of bio-terrorism directed against food and agriculture (Monke, 2004). Rapid detection techniques for bioweapon agents are a critical need for the first-responder community. Among the nucleic acid-based diagnostic techniques, those involving the Polymerase Chain Reaction (PCR; Mullis and Faloona, 1987) are the most suited for early detection of phytopathogenic agents, due to their high sensitivity and the potential for automation. Many sequence source types could be selected and used as target for specific primer design. These may include, for instance, Random Amplified Polymorphic DNAs (Williams et al., 1990; Welsh and McClelland, 1990), internal transcribed spacer (ITS) regions of the ribosomal RNA genes (White et al., 1990) or other specific gene sequences. Primer sets can be designed to target specificity at the genus, species, or physiological race level, to distinguish a particular pathogen from closely related organisms. A common and tedious task for researchers and technicians is to search for and retrieve bibliographic references of published and validated specific primer sets for a given pathogen querying the Internet, abstract collections and monthly journals’ tables of contents. Very few examples of specific primer set collections for phytopathogenic agents have been released: a summary of primers for the diagnostic characterization of phytopathogenic bacteria seems to be the only one printed so far (Louws et al., 1999). Moreover, among 719 molecular biology databases publicly available recorded by Galperin (2006) or among the 2470 BMC biomedical databases catalog available at http://databases.biomedcentral.com/, no online repository of primer sets of this kind is accessible. To overcome this lack of information, we released the first online searchable database of primer sets useful for the detection and identification of plant pathogenic fungi

Open access e valutazione della ricerca scientifica

La relazione affronta il problema della valutazione della ricerca scientifica, dell'IF (Impact Factor) per valutare gli autori, della citation impact e gli effetti dell'Open Access sulla incisività della ricerca nella comunità scientifica e nella società

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

Plant extracts as biocontrol agents against Aspergillus carbonarius growth and ochratoxin A production in grapes

Aspergillus carbonarius (Bainier) Thom. is an important pathogen and ochratoxin A (OTA) producer in grapes that can be controlled by adopting sustainable approaches. Here we evaluate the application of natural plant extracts as an alternative to synthetic fungicides to reduce OTA contamination and to prevent infection of grapes by two isolates of A. carbonarius. In a preliminary screening, natural extracts of chestnut flower, cistus, eucalyptus, fennel, and orange peel were evaluated for their antifungal and anti-mycotoxigenic efficiency in a grape-based medium at concentrations of 10 and 20 mg/mL. Cistus and orange peel extracts demonstrated the best anti- fungal activity at both concentrations. Although the eucalyptus extract demonstrated no significant effect on Aspergillus vegetative growth, it significantly reduced OTA by up to 85.75 % at 10 mg/mL compared to the control. Chestnut flower, cistus, eucalyptus, and orange peel extracts were then tested at the lowest concen- tration (10 mg/mL) for their antifungal activity in artificially inoculated grape berries. The cistus and orange peel extracts demonstrated the greatest antifungal activity and significantly reduced mold symptoms in grapes. Moreover, all tested natural extracts were able to reduce OTA content in grape berries (17.7 ± 8.3 % - 82.3 ± 3.85 % inhibition), although not always significantly. Eucalyptus extract was particularly efficient, inhibiting OTA production by both strains of A. carbonarius by up to >80 % with no effects on fungal growth. The use of natural eucalyptus extract represents a feasible strategy to reduce OTA formation without disrupting fungal growth, apparently maintaining the natural microbial balance, while cistus and orange peel extracts appear promising as inhibitors of A. carbonarius mycelial growth. Our findings suggest that plant extracts may be useful sources of bioactive chemicals for preventing A. carbonarius contamination and OTA production. Nonetheless, it will be necessary to evaluate their effect on the organoleptic properties of the grapes.This work was supported by the Foundation for Science and Tech- nology (FCT, Portugal) #1 under Grant from national funds FCT/MCTES (PIDDAC) to CIMO [number UIDB/00690/2020 and UIDP/00690/ 2020] and SusTEC [number LA/P/0007/2020]; the European Regional Development Fund (ERDF) through the Competitiveness and Interna- tionalization Operational Program (CIOP) #2 under Grant dedicated to the project “PreVineGrape - Development of a biofungicide to combat grapevine diseases [number POCI-01-0247-FEDER-049695].info:eu-repo/semantics/publishedVersio

Electrophoretic karyotype variation among pathotypes of Fusarium oxysporum f.sp. dianthi

Karyotype analysis by pulsed-field gel electrophoresis was applied to characterize isolates of Fusarium oxysporum f.sp. dianthi, the causal agent of Fusarium wilt on carnation. Eleven distinct chromosomal DNA patterns were detected among 38 pathogenic isolates, and the total genome size was estimated to range from 23·7 to 36·4 Mb. Except for isolates belonging to pathotypes 2 and 4, all members of the same pathotype shared overlapping electrophoretic karyotypes. Karyotypes of isolates assigned to pathotypes 1 and 8 showed a high degree of similarity, in accordance with VCG and RFLP analysis. The same electrophoretic karyotype was also shared by members of pathotypes 2 and 5, thus confirming results obtained by both VCG and RFLP grouping, A single representative of pathotype 6, previously confined to the same VCG and RFLP group as pathotypes 2 and 5, had a slightly different chromosomal pattern. Isolates assigned to pathotype 4 showed four related karyotypes which partially differed in both the number and size of chromosomal bands. However, all strains assigned to this pathotype shared a basic profile of nine chromosomal bands, while two low-molecular-weight bands were present or absent. The findings are discussed with regard both to the suitability of race distinction in the case of the special form dianthi of F. oxysporum and to the use of karyotype analysis by PFGE as a tool for the study of the population genetics of this fungu

PCR detection of Fusarium oxysporum f. sp. basilici on basil

Sixty-nine amplified DNA fragments, generated from different isolates of Fusarium oxysporum f. sp. basilici, were tested for F. oxysporum f. sp. basilici–specificity in a dot blot assay. One 1,038-bp fragment hybridized to DNA from all F. oxysporum f. sp. basilici isolates but not to DNA obtained from F. oxysporum isolates nonpathogenic to basil or representatives of other formae speciales of F. oxysporum, or from isolates of F. redolens, F. tabacinum, Rhizoctonia solani, Sclerotinia sclerotiorum, S. minor, and Pythium ultimum obtained from diseased basil. This fragment was cloned and sequenced, and three pairs of F. oxysporum f. sp. basilici– specific primers were designed, giving rise to amplification products of 943, 382, and 330 bp. A nested PCR assay allowed detection of F. oxysporum f. sp. basilici in diseased seedlings and in artificially and naturally contaminated seeds. The theoretical detection limit of this system was 102 fungal propagules per 100 seeds on artificially contaminated samples, while on naturally contaminated commercial seed lots, 32 propagules per 100 seeds were detected

A <i>Saccharomyces cerevisiae</i> wine strain inhibits growth and decreases ochratoxin a biosynthesis by <i>Aspergillus carbonarius</i> and <i>Aspergillus ochraceus</i>

The aim of this study was to select wine yeast strains as biocontrol agents against fungal contaminants responsible for the accumulation of ochratoxin A (OTA) in grape and wine and to dissect the mechanism of OTA detoxification by a Saccharomyces cerevisiae strain (DISAABA1182), which had previously been reported to reduce OTA in a synthetic must. All of the yeast strains tested displayed an ability to inhibit the growth of Aspergillus carbonarius both in vivo and in vitro and addition of culture filtrates from the tested isolates led to complete inhibition of OTA production. S. cerevisiae DISAABA1182 was selected and further tested for its capacity to inhibit OTA production and pks (polyketide synthase) transcription in A. carbonarius and Aspergillus ochraceus in vitro. In order to dissect the mechanism of OTA detoxification, each of these two fungi was co-cultured with living yeast cells exposed to yeast crude or to autoclaved supernatant: S. cerevisiae DISAABA1182 was found to inhibit mycelial growth and OTA production in both Aspergilli when co-cultured in the OTA-inducing YES medium. Moreover, a decrease in pks transcription was observed in the presence of living cells of S. cerevisiae DISAABA1182 or its supernatant, while no effects were observed on transcription of either of the constitutively expressed calmodulin and β-tubulin genes. This suggests that transcriptional regulation of OTA biosynthetic genes takes place during the interaction between DISAABA1182 and OTA-producing Aspergilli

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

Chemically modified β-cyclodextrins useful in developing biosensors of agricultural and food relevance

β-cyclodextrin (β-CD), a natural, non-toxic cycloeptaamilose macrocycle, is a useful biomatrix for immobilizing enzymes on a biosensor surface because of the affinity of its cavity for hydrophobic guest molecules (e.g., aminoacids). In this work β-CD has been successfully modified with different poly-carboxylic acids (PCAs) including 1,2,3,4-butanetetracarboxylic acid. Time activation, pH, pressure and stoichiometry were optimized in order to achieve selected substitutions on the macrocycle hydroxy groups. The modified β-CDs, prepared under mild conditions, are completely water-soluble and could be grafted on a biosensor surface

Transformants of <i>Trichoderma longibrachiatum</i> overexpressing the beta-1,4-endoglucanase gene <i>egl1</i> show enhanced biocontrol of <i>Pythium ultimum</i> on cucumber

Nine transformants of Trichoderma longibrachiatum with extra copies of the egl1 gene were studied for mitotic stability, endoglucanase production, and biocontrol activity against Pythium ultimum on cucumber seedlings. The transformants showed a significantly higher level of expression of the egl1 gene in comparison to the wild type under both inducing and noninducing growth conditions. Transformants with the egl1 gene under the control of a constitutive promoter had the highest enzymatic activity. Both the endoglucanase activity and the transforming sequences were stable under nonselective conditions. When applied to cucumber seeds sown in P. ultimum-infested soil, T. longibrachiatum transformants with increased inducible or constitutive egl1 expression generally were more suppressive than the wild-type strain