Evaluation of in-vitro methods to select effective streptomycetes against toxigenic fusaria

Biocontrol microorganisms are emerging as an effective alternative to pesticides. Ideally, biocontrol agents (BCAs) for the control of fungal plant pathogens should be selected by an in vitro method that is high-throughput and is predictive of in planta efficacy, possibly considering environmental factors, and the natural diversity of the pathogen. The purpose of our study was (1) to assess the effects of Fusarium strain diversity (N = 5) and culture media (N = 6) on the identification of biological control activity of Streptomyces strains (N = 20) against Fusarium pathogens of wheat in vitro and (2) to verify the ability of our in vitro screening methods to simulate the activity in planta. Our results indicate that culture media, Fusarium strain diversity, and their interactions affect the results of an in vitro selection by dual culture assay. The results obtained on the wheat-based culture media resulted in the highest correlation score (r = 0.5) with the in planta root rot (RR) inhibition, suggesting that this in vitro method was the best predictor of in planta performance of streptomycetes against Fusarium RR of wheat assessed as extension of the necrosis on the root. Contrarily, none of the in vitro plate assays using the media tested could appropriately predict the activity of the streptomycetes against Fusarium foot rot symptoms estimated as the necrosis at the crown level. Considering overall data of correlation, the activity in planta cannot be effectively predicted by dual culture plate studies, therefore improved in vitro methods are needed to better mimic the activity of biocontrol strains in natural conditions. This work contributes to setting up laboratory standards for preliminary screening assays of Streptomyces BCAs against fungal pathogens

Genetic Similarity of Flag Shoot and Ascospore Subpopulations of Erysiphe necator in Italy

The overwintering mode of the grape powdery mildew fungus, Erysiphe necator (syn. Uncinula necator), as mycelium in dormant buds (resulting in symptoms known as flag shoots) or as ascospores in cleistothecia, affects the temporal dynamics of epidemics early in the growing season. We tested whether distinct genetic groups (I and III) identified previously in E. necator correlate to overwintering modes in two vineyards in Tuscany, Italy, to determine whether diagnostic genetic markers could be used to predict overwintering. Samples from one vineyard were collected from flag shoots; the other vineyard, 60 km away, had no flag shoots, and mildew colonies were assumed to be derived from ascospores. Genetic markers putatively diagnostic for groups I and III showed that both types were common in the flag shoot subpopulation. Both genetic types were found in the ascospore population, although group III was dominant. We did not find strong genetic differentiation between the two subpopulations based on inter-simple sequence repeat markers. Although there was significant (P < 0.001) genetic differentiation between these subpopulations in 1997 and when 1997 and 1998 subpopulations were pooled (θ = 0.214 and 0.150, respectively), no differentiation was evident between vineyards in 1998 (θ = 0.138, P = 0.872). Moreover, we did not observe distinct lineages corresponding to overwintering modes, as observed in previous studies. We could not determine if differentiation resulted from biological differences or restricted gene flow between the two vineyards. Our samples were taken from both subpopulations early in the epidemic, while previous studies confounded overwintering mode and sampling time. These results do not support a strong correlation between overwintering and genetic groups, highlighting the need to base population biology studies on sound biological and epidemiological knowledge

Erythrodermic psoriasis in a dialyzed patient successfully treated with Secukinumab

Erythrodermic psoriasis is a severe, life-threatening condition with additional complications, when occurring in hemodialyzed patients, as the majority of treatments are contraindicated. A 44-years-old man, of Philippine origins, with a 15-years-history of psoriasis treated with cyclosporine developed progressive hypertension and renal insufficiency. Despite drug dismission, renal function worsen to end-stage, and hemodialysis was necessary three times a week. Phototherapy was not able to control the skin condition, progressing to erythroderma, and after nephrology consultation, the patient consent to the off-label secukinumab treatment, at the standard regimen (300 mg subcutaneously once weekly at weeks 0-4 followed by 300 mg every 4 weeks). Seven days after the first injection, a rapid improvement was noted, with the psoriasis area severity index (PASI) score passing from 31.5 to 17.6. At the 52-week-follow-up visit, the patient was completely clarified, without any side effects. The case supports secukinumab effectiveness and safety in difficult patients, including erythrodermic psoriasis with end-stage renal failure, as drug plasma levels seem not to be affected by hemodialysis. Results are rapidly achieved, and long term maintained, with the additional advantage of a very comfortable monthly administration

Grapevine Powdery Mildew: Fungicides for Its Management and Advances in Molecular Detection of Markers Associated with Resistance

Grapevine powdery mildew is a principal fungal disease of grapevine worldwide. Even though it usually does not cause plant death directly, heavy infections can lead to extensive yield losses, and even low levels of the disease can negatively affect the quality of the wine. Therefore, intensive spraying programs are commonly applied to control the disease, which often leads to the emergence and spread of powdery mildew strains resistant to different fungicides. In this review, we describe major fungicide classes used for grapevine powdery mildew management and the most common single nucleotide mutations in target genes known to confer resistance to different classes of fungicides. We searched the current literature to review the development of novel molecular methods for quick detection and monitoring of resistance to commonly used single-site fungicides against Erysiphe necator. We analyze and compare the developed methods. From our investigation it became evident that this research topic has been strongly neglected and we hope that effective molecular methods will be developed also for resistance monitoring in biotroph pathogens

Selection of Streptomyces against soil borne fungal pathogens by a standardized dual culture assay and evaluation of their effects on seed germination and plant growth

Background: In the search for new natural resources for crop protection, streptomycetes are gaining interest in agriculture as plant growth promoting bacteria and/or biological control agents. Because of their peculiar life cycle, in which the production of secondary metabolites is synchronized with the development of aerial hyphae and sporulation, the commonly used methods to screen for bacterial antagonists need to be adapted. Results: The dual culture assay was standardized in terms of inoculation timing of Streptomyces antagonist and pathogen, and growth rate of different fungal pathogens. In case of fast-growing fungi, inoculation of the antagonist 2 or 3 days prior to the pathogen resulted in significantly stronger inhibition of mycelium growth. One hundred and thirty Streptomyces strains were evaluated against six destructive soil borne pathogens. The activity of strains varied from broad-spectrum to highly specific inhibition of individual pathogens. All strains inhibited at least one tested pathogen. Three strains, which combined the largest broad-spectrum with the highest inhibition activity, were selected for further characterization with four vegetable species. All of them were able to colonize seed surface of all tested vegetable crops. They mostly improved radicle and hypocotyl growth in vitro, although no statistically significant enhancement of biomass weight was observed in vivo. Occasionally, transient negative effects on germination and plant growth were observed. Conclusions: The adapted dual culture assay allowed us to compare the inhibition of individual Streptomyces strains against six fungal soil borne pathogens. The best selected strains were able to colonize the four vegetable crops and have a potential to be developed into biocontrol products. Although they occasionally negatively influenced plant growth, these effects did not persist during the further development. Additional in vivo studies are needed to confirm their potential as biological control or plant growth promoting agents

Additional file 1: of Selection of Streptomyces against soil borne fungal pathogens by a standardized dual culture assay and evaluation of their effects on seed germination and plant growth

Table S1. Mycelial growth inhibition of six soil borne pathogens by 130 Streptomyces strains. (DOCX 36 kb

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Stilbenoids as Antifungals to Counteract Rice Blast Pathogen Pyricularia oryzae

Fungi are among the greatest biotic threats to agricultural and food security. Intensive monoculture cropping and resistance to single-site fungicides in plant pathogens urge the discovery and development of novel compounds that possibly interfere with essential cellular processes in multiple ways. In this article, we describe our recent efforts addressed to the identification of natural compounds as multitarget biofungicides. A set of natural monomeric and dimeric compounds belonging to the class of stilbenoids were synthesized and tested against wild-type (WT) and strobilurin-resistant (RES) strains of Pyricularia oryzae, one of the most dangerous fungal phytopathogens. Monomers deoxyrhapontigenin, pinostilbene, and DMHS showed inhibitory activity higher than 40%, with deoxyrhapontigenin having the highest activity on mycelial growth (60–80% inhibition) on both WT and RES P. oryzae strains. Furthermore, we designed and synthesized a set of molecules having a nature-derived stilbene fragment merged with the pharmacophoric portion of strobilurins, namely, a β-methoxyacrylate moiety. We identified two molecules with activity comparable to the reference commercial fungicide azoxystrobin. However, low mycelium growth inhibition of resistant strains indicates that these compounds most likely retain the strobilurin-like mechanism of action. Overall, the results suggest that natural stilbenoids might be used as environmentally friendly biofungicides in rice blast management

SMAD 8 binding to mice Msx1 basal promoter is required for transcriptional activation

The Msx1 gene in mice has been proven to be induced by BMP (bone morphogenetic protein) proteins, and three binding sites for SMAD, an intracellular BMP signalling transducer, have already been identified in its promoter. Gel shift analyses were performed and they demonstrated that the consensus found very near the transcription start site, a region designed BP (basal promoter), is functional for binding nuclear proteins from 10.5, 11.5 and 13.5 dpc (days post-coitum) embryos. Notably, this binding occurs only when the SMAD-binding consensus sequence is maintained, suggesting that it is required for the formation of a protein complex over BP. Binding of purified SMAD 1 and SMAD 4 as well as supershift assay with SMAD 1/SMAD 5/SMAD 8 antibody proved that a SMAD protein is present in this complex. Transfection assays in cell cultures with fragments from BP driving the expression of luciferase confirmed that only in the presence of the SMAD consensus site is Msx1 expression activated. A proteomic analysis of the complex components after immunoprecipitation identified several proteins necessary to activate transcription including SMAD 8. Our results suggest that BMP2/BMP4 signalling through SMAD 8 is required for transcriptional activation of the mouse Msx1 gene