Critical assessment of Streptomyces spp. able to control toxigenic fusaria in cereals : a literature and patent review

Mycotoxins produced by Fusarium species on cereals represent a major concern for food safetyworldwide. Fusariumtoxinsthatarecurrentlyunderregulationfortheircontentinfoodinclude trichothecenes, fumonisins, and zearalenone. Biological control of Fusarium spp. has been widely explored with the aim of limiting disease occurrence, but few e\ufb00orts have focused so far on limiting toxin accumulation in grains. The bacterial genus Streptomyces is responsible for the production of numerous drug molecules and represents a huge resource for the discovery of new molecules. Streptomycesspp. arealsoe\ufb03cientplantcolonizersandabletoemploydi\ufb00erentmechanismsofcontrol against toxigenic fungi on cereals. This review describes the outcomes of research using Streptomyces strains and/or their derived molecules to limit toxin production and/or contamination of Fusarium species in cereals. Both the scienti\ufb01c and patent literature were analyzed, starting from the year 2000, and we highlight promising results as well as the current pitfalls and limitations of this approach

Selection of an Endophytic Streptomyces sp. Strain DEF09 From Wheat Roots as a Biocontrol Agent Against Fusarium graminearum

Selection of biological control agents (BCA) profits from an integrated study of the tripartite interactions occurring among the BCA, the plant and the pathogen. The environment plays a crucial role in the efficacy of BCA, therefore, the selection process shall utmost mimic naturally occurring conditions. To identify effective biocontrol strains against Fusarium graminearum, the major cause of Fusarium head blight (FHB) in wheat and deoxynivalenol (DON) accumulation in grains, a workflow consisting of in vitro and in vivo assays was set up. Twenty-one Streptomyces strains, 16 of which were endophytes of different plants, were analyzed. In vitro and in vivo tests characterized their plant growth promoting (PGP) traits. Biocontrol activity against F. graminearum was firstly assessed with a dual culture assay. An in vivo germination blotter assay measured Fusarium foot rot and root rot symptoms (FFR-FRR) reduction as well as growth parameters of the plant treated with the Streptomyces strains. A selected subset of Streptomyces spp. strains was then assessed in a growth chamber measuring FFR symptoms and growth parameters of the wheat plant. The approach led to the identification of an effective Streptomyces sp. strain, DEF09, able to inhibit FHB on wheat in controlled conditions by blocking the spread of the pathogen at the infection site. The results were further confirmed in field conditions on both bread and durum wheat, where DEF09 decreased disease severity up to 60%. This work confirms that FRR and FFR pathosystems can be used to identify BCA effective against FHB

Fungal biofilms: targets for the development of novel strategies in plant disease management

The global food supply has been facing increasing challenges during the first decades of the 21st century. Disease in plants is an important constraint to worldwide crop production, accounting for 20\u201340% of its annual harvest loss. Although the use of resistant varieties, good water management and agronomic practices are valid management tools in counteracting plant diseases, there are still many pathosystems where fungicides are widely used for disease management. However, restrictive regulations and increasing concern regarding the risk to human health and the environment, along with the incidence of fungicide resistance, have discouraged their use and have prompted for a search for new efficient, ecologically friendly and sustainable disease management strategies. The recent evidence of biofilm formation by fungal phytopathogens provides the scientific framework for designing and adapting methods and concepts developed by biofilm research that could be integrated in IPM practices. In this perspective paper, we provide evidence to support the view that the biofilm lifestyle plays a critical role in the pathogenesis of plant diseases. We describe the main factors limiting the durability of single-site fungicides, and we assemble the current knowledge on pesticide resistance in the specific context of the biofilm lifestyle. Finally, we illustrate the potential of antibiofilm compounds at sub-lethal concentrations for the development of an innovative, eco-sustainable strategy to counteract phytopathogenic fungi. Such fungicide-free solutions will be instrumental in reducing disease severity, and will permit more prudent use of fungicides decreasing thus the selection of resistant forms and safeguarding the environment

New formulation and delivery method of Cryphonectria parasitica for biological control of chestnut blight

Aims: This study aimed to develop a new formulation of Cryphonectria parasitica hypovirulent mycelium suitable for inoculations of tall trees from the ground. C. parasitica hypovirulent strains are widely used for biological control of chestnut blight. However, it is often inconsistent and ineffective not only for biological reasons, but also because the current manual application of hypovirulent strains on adult plants is difficult, time-consuming and expensive. Here, we propose an improved formulation and more effective mode of application of hypovirulent strains, which could boost chestnut blight biocontrol. Methods and results: The Cp 4.2H hypovirulent strain was formulated as mycelium disks with polyethylene glycol (PEG) and hydroxypropyl methylcellulose (HPMC), loaded into lead-free pellets used as carriers to inoculate cankers on chestnut stems by shooting. The formulation of the mycelium did not hamper its viability which was stable, with an estimated shelf life of 72 days at 6\ub11 \ub0C. The inoculum effectiveness was confirmed ex planta and in planta in a small-scale pilot study in field, where formulated mycelium disks of hypovirulent strain Cp 4.2H were airgun shot into the chestnut bark. In planta, Cp 4.2H was recovered in 37% of bark samples taken around the inoculated points one year after the treatment. Conclusions: We demonstrated that the proposed airgun shooting inoculation method of C. parasitica hypovirulent strain formulated as mycelium disks is suitable for treatment of adult chestnut trees. Significance and impact of the study: The proposed method could be a valid alternative to the traditional manual technique of chestnut biocontrol. The main advantages are the cost-effectiveness and the ease to treat high-positioned, otherwise unreachable cankers both in orchards and forests. This article is protected by copyright. All rights reserved

Investigating Useful Properties of Four Streptomyces Strains Active against Fusarium graminearum Growth and Deoxynivalenol Production on Wheat Grains by qPCR

Streptomyces spp. can be exploited as biocontrol agents (BCAs) against plant pathogens such as Fusarium graminearum, the main causal agent of Fusarium head blight (FHB) and against the contamination of grains with deoxynivalenol (DON). In the present research, four Streptomyces strains active against F. graminearum in dual plate assays were characterized for their ability to colonize detached wheat grains in the presence of F. graminearum and to limit DON production. The pathogen and BCA abundance were assessed by a quantitative real-time PCR, while DON production was assessed by HPLC quantification and compared to ergosterol to correlate the toxin production to the amount of fungal mycelium. Fungal growth and mycotoxin production were assessed with both co-inoculation and late inoculation of the BCAs in vitro (three days post-Fusarium inoculation) to test the interaction between the fungus and the bacteria. The level of inhibition of the pathogen and the toxin production were strain-specific. Overall, a higher level of DON inhibition (up to 99%) and a strong reduction in fungal biomass (up to 71%) were achieved when streptomycetes were co-inoculated with the fungus. This research enabled studying the antifungal efficacy of the four Streptomyces strains and monitoring their development in DON-inducing conditions

Use of Streptomyces spp. as biocontrol agents of wheat crown rot caused by fusaria

Crown rot of wheat is caused mainly by Fusarium species and in specific climatic conditions can reduce production up to 50-90%. Biocontrol methods are an interesting approach to satisfy the increasing demand for alternatives to chemical fungicides. Streptomyces spp. act as growth promoters as well as biocontrol agents. Therefore, they are promising candidates for seed treatments, which combine their ability to produce a wide range of biologically active molecules with the potential to establish tight interactions with plants. Forty-seven Streptomyces spp. strains were selected among 1500, isolated from roots or rhizosphere of various crops. Their ability to reduce mycelial growth (up to 80%) of five Fusarium graminearum and F. culmorum strains (N=5), representing geographical, genotypic and toxigenic diversity, was determined in vitro. Streptomycetes showing inhibition >20% were further tested in vitro on wheat seedlings (cv. Bandera) for their ability to reduce disease symptoms. The root-necrosis was reduced up to 50% after 4 dpi. Moreover, the strains reduced the severity of crown rot up to 80% after 6 dpi in comparison to the untreated control. The selected strains did not inhibit the growth of the seedlings at the tested condition. Further in vivo studies are foreseen to assess their plant growth promoting ability. Until now, our results confirm that streptomycetes are a promising source for developing organic seed treatments

Phenotypic and genotypic diversity in Ciborinia camelliae Kohn isolates from Italy

Ciborinia camelliae, causal agent of Camellia flower blight, belongs to Ascomycota, family Sclerotiniaceae. The pathogen shows host and organ specificity infecting only flowers of camellias, causing a serious damage not only to the ornamental component of the plant, but interfering with the fruit development reducing thus the seed yield and oil production. Interestingly, ascomata of Ciborinia show an anti-feeding effect on collembola, suggesting its importance for the production of novel bioactive compounds. From Japan the pathogen spread worldwide quickly and despite it has been reported in all camellia-cultivated areas few studies have been carried out from a mycological and population variability point of view. Thirty strains from five Italian regions were characterized on four different culture media at six temperatures, describing their development daily up to 14 days. Based on these data 10 different morphotypes of C. camelliae were characterized. Multigene sequencing and universally primed PCR technique were used to assess genetic variability among C. camelliae isolates. The genotypic approach confirmed the phenotypic variability among the Italian strains compared to the results of other research groups. Further studies are therefore needed in this sector considering also the geographical distribution of the pathogen. Finally, exploiting fungal variability found in our samples may lead to the identification of strains able to produce high levels of bioactive molecules against insect pests or microorganisms

Development of a Fusarium graminearum biosensor assay to monitor the activity of naturally derived products to control trichothecene production

In the present study, we developed a microplate reader assay to detect natural products able to limit toxin production by the fungus Fusarium graminearum, the major cause of Fusarium Head Blight in cereals and trichothecene contamination in grains worldwide. In the biosensor assay, we exploited a F. graminearum isolate expressing GFP-tagged trichodiene synthase, encoded by TRI5 (TRI5-GFP), which catalyses the first step of the trichothecene biosynthesis pathway. This allowed us to monitor the first step of toxin production by fluorimetric assay. Fungal spores were treated with filtrates from streptomycete liquid cultures in order to evaluate their influence on fungal growth and toxin production, integrating absorbance and fluorescence measurements. The correlation between the fluorimetric assay based on the TRI5-GFP detection and the amount of deoxynivalenol (DON) measured with ELISA technique was assessed. The new method allowed to identify crude filtrates able to reduce toxin production as accurately as ELISA. The main advantages of the newly developed assay are the reduction of the analysis costs and lower use of chemicals and consumables. It contributes to increase the speed and capacity to screen large set of molecules and natural extracts. A historical collection of Streptomyces strains was screened by the newly developed method and five bacterial strains have been identified as producing bioactive molecules able to inhibit the fungal growth and/or the toxin synthesis (DON)

Kinetic and continuum modeling of high-temperature oxygen and nitrogen binary mixtures

The present paper provides a comprehensive comparative analysis of thermochemistry models of various fidelity levels developed in leading research groups around the world. Fully kinetic, hybrid kinetic-continuum, and fully continuum approaches are applied to analyze parameters of hypersonic flows starting from the revision of single-temperature rate constants up to the application in 1-D postshock conditions. Comparison of state-specific and two-temperature approaches shows there are very significant and often qualitative differences in the time-dependent nonequilibrium reaction rates and their ratio to the corresponding single-temperature rates. A major impact of the vibration-dissociation coupling on the temporal relaxation of gas properties is shown. For instance, the legacy Park's model has a strongly nonlinear behavior of nonequilibrium reaction rate with vibrational temperature, while a nearly linear shape exists for all state-specific approaches. Analysis of vibrational level populations in the nonequilibrium region shows a profound impact of the numerical approach and the model on the population ratios, and thus vibrational temperatures inferred from such ratios. The difference in the ultraviolet absorption coefficients, calculated by a temperature-based spectral code using vibrational populations from state-specific and kinetic approaches, is found to exceed an order of magnitude

Kinetic and continuum modeling of high-temperature air relaxation

Fully kinetic, vibrationally kinetic, and continuum solvers with varying model fidelity are used in this work to model the high-temperature relaxation of air in 7230 and 15,000 K adiabatic heat baths and a 6 km/s hypersonic flow over a cylinder. The results show significant impact of uncertainties in vibrational relaxation times and reaction rate constants on thermal and chemical relaxation, in particular, on gas temperature and species mole fractions. Most notably, these uncertainties need to be reduced for collisions that include nitric oxide. Order-of-magnitude differences in the nitric oxide dissociation and recombination rates have a large impact on the peak NO mole fraction immediately behind the shock and surface-distributed heat flux, respectively. High-fidelity kinetic and continuum approaches are found to have different reaction channels having the largest effect on species mole fractions and gas temperature: N-2+O exchange and O-2+O dissociation in the former, and NO+O and O-2+N-2 dissociation in the latter