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Phytoseiid predatory mites are one of the most important groups of biocontrol agents, commonly used in biological control. The ability to produce these predatory mites economically, at high density on cheap factitious food sources, is a major contributor to their success. Astigmatid mites are the most widely used factitious food for this purpose. In this study, we investigated the potential application of the leaf-dwelling astigmatid mite Czenspinskia transversostriata (Oudemans) (Acari: Winterschmidtiidae) as a prey mite in biological control. We tested whether C. transversostriata is a suitable food source for the predatory mite Amblyseius swirskii Athias-Henriot (Acari: Phytoseiidae), both in the laboratory and on cucumber plants. Based on a reproduction trial, C. transversostriata proved to be an equally good food source compared to both pollen of Typha angustifolia L. (Poales: Typhaceae) and a frequently used prey mite Carpoglyphus lactis L. (Acari: Carpoglyphidae). In a pre-establishment trial on cucumber plants, populations of A. swirskii reached equally high densities when supplemented with C. transversostriata, compared to C. lactis. Lastly, we show that C. transversostriata is capable of feeding and reproducing on powdery mildew growing on cucumber plants, thereby slowing down the development of the pathogenic fungus. Results derived from this study show that C. transversostriata may have multiple potential applications in biological control programs.

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Fungal control strategies based on the use of Bacillus have emerged in agriculture as eco-friendly alternatives to replace/reduce the use of synthetic pesticides. Bacillus sp. P1 was reported as a new promising strain for control of Aspergillus carbonarius, a known producer of ochratoxin A, categorized as possible human carcinogen with high nephrotoxic potential. Grape quality can be influenced by vineyard management practices, including the use of fungal control agents. The aim of this study was to evaluate, for the first time, the quality parameters of Chardonnay grapes exposed to an antifungal Bacillus-based strategy for control of A. carbonarius, supporting findings by genomic investigations. Furthermore, genomic tools were used to confirm that the strain P1 belongs to the non-pathogenic species Bacillus velezensis and also to certify its biosafety. The genome of B. velezensis P1 harbors genes that are putatively involved in the production of volatiles and hydrolytic enzymes, which are responsible for releasing the free form of aroma compounds. In addition to promote biocontrol of phytopathogenic fungi and ochratoxins, the treatment with B. velezensis P1 did not change the texture (hardness and firmness), color and pH of the grapes. Heat map and hierarchical clustering analysis (HCA) of volatiles evaluated by GC/MS revealed that Bacillus-treated grapes showed higher levels of compounds with a pleasant odor descriptions such as 3-hydroxy-2-butanone, 2,3-butanediol, 3-methyl-1-butanol, 3,4-dihydro-ß-ionone, ß-ionone, dihydroactinidiolide, linalool oxide, and ß-terpineol. The results of this study indicate that B. velezensis P1 presents desirable properties to be used as a biocontrol agent.

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Antifungal peptides (AFPs) can be used as novel preservatives, but achieving large-scale production and application remains a long-term challenge. In this study, we developed a hybrid peptide MD (metchnikowin-drosomycin fusion) secreted into Escherichia coli supernatant, demonstrating strong inhibitory activity against Aspergillus flavus and Botrytis cinerea. The fusion tag did not impact its activity. Moreover, an endotoxin-free and oxidative leaky strain was developed by knocking out the trxB, gor, and lpp genes of endotoxin-free E. coli ClearColi-BL21(DE3). This strain facilitates the proper folding of multi-disulfide bond proteins and promotes the extracellular production of recombinant bioactive AFP MD, achieving efficient production of endotoxin-free MD. In addition, temperature control replaces chemical inducers to further reduce production costs and circumvent the toxicity of inducers. This extracellularly produced MD exhibited favorable effectiveness in inhibiting fruit mold growth, and its safety was preliminarily established by gavage testing in mice, suggesting that it can be developed into a green and sustainable fruit fungicide. In conclusion, this study provides novel approaches and systematic concepts for producing extracellularly active proteins or peptides with industrial significance. KEY POINTS: • First report of extracellular production of bioactive antifungal peptide in Escherichia coli. • The hybrid antifungal peptide MD showed strong inhibitory activity against Aspergillus flavus and Botrytis cinerea, and the activity was not affected by the fusion tag. • Endotoxin-free oxidative Escherichia coli suitable for the expression of multi-disulfide bond proteins was constructed.

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The use of agrochemicals has caused environmental problems and toxicity to humans, so natural alternatives for disease control during harvest and postharvest have been evaluated. The aim of this study was to evaluate cinnamon essential oil, neem oil, and black sapote fruit extract for in vitro inhibition of fungi isolated from chayote fruit. The extracts were applied at 300, 350, and 400 ppm in Petri dishes and the mycelial growth of Fusarium oxysporum, Fusarium solani, Goetrichum sp., and Phytophthora capsici was evaluated for 7 days, and the percentage of mycelial growth inhibition per day was calculated. Cinnamon oil showed a fungicidal effect at all concentrations. Neem oil at 400 ppm showed a 42.3% reduction in the growth of F. solani and 27.8% reduction in the growth of F. oxysporum, while at 350 ppm it inhibited the mycelial growth of Phytophthora capsici by 53.3% and of Goetrichum sp. by 20.9%; finally, the black sapote extract at 400 ppm inhibited 21.9-28.6% of the growth of all fungi. The growth of postharvest fungi on chayote fruit could be prevented or reduced by applying the plant extracts evaluated at adequate concentrations.

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Maize is a significant crop to the global economy and a key component of food and feed, although grains and whole plants can often be contaminated with mycotoxins resulting in a general exposure of the population and animals. To investigate strategies for mycotoxins control at the grain production level, a pilot study and exploratory research were conducted in 2019 and 2020 to compare levels of mycotoxins in grains of plants treated with two fertilizers, F-BAC and Nefusoil, under real agricultural environment. The 1650 grains selected from the 33 samples were assessed for the presence of both Fusarium species and mycotoxins. Only fumonisins and deoxynivalenol were detected. Fumonisin B1 ranged from 0 to 2808.4 µg/Kg, and fumonisin B2 from 0 to 1041.9 µg/Kg, while deoxynivalenol variated from 0 to 465.8 µg/Kg. Nefusoil showed to be promising in regard to fumonisin control. Concerning the control of fungal contamination rate and the diversity of Fusarium species, no significant differences were found between the two treatments in any of the years. However, a tendency for was observed Nefusoil of lower values, probably due to the guaranteed less stressful conditions to the Fusarium spp. present in the soil, which do not stimulate their fumonisins production.

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Aspergillus flavus is an important pathogenic fungus affecting many crops and is one of the main sources of their aflatoxin contamination. The primary method of limiting this pathogen is using chemical fungicides, but researchers focus on searching for other effective agents for its control due to many disadvantages and limitations of these agrochemicals. The results obtained in the present study indicate the high potential of two yeast strains, Aureobasidium pullulans PP4 and A. pullulans ZD1, in the biological control of A. flavus. Under in vitro conditions, mycelial growth was reduced by 53.61% and 63.05%, and spore germination was inhibited by 68.97% and 79.66% by ZD1 and PP4 strains, respectively. Both strains produced the lytic enzymes chitinase and ß-1,3-glucanase after 5 days of cultivation with cell wall preparations (CWP) of A. flavus in the medium as a carbon source. In addition, the tested yeasts showed the ability to grow over a wide range of temperatures (4-30 °C), pH (4-11), and salinity (0-12%) and showed tolerance to fungicides at concentrations corresponding to field conditions. Both isolates tested were highly tolerant to cupric oxychloride, showing biomass gains of 85.84% (ZD1) and 87.25% (PP4). Biomass growth in the presence of fungicides azoxystrobin was 78.71% (ZD1) and 82.65% (PP4), while in the presence of difenoconazole, it was 70.09% (ZD1) and 75.25% (PP4). The yeast strains were also tested for antagonistic effects against A. flavus directly on tomato fruit. Both isolates acted effectively by reducing lesion diameter from 29.13 mm (control) to 8.04 mm (PP4) and 8.83 mm (ZD1).

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Efficient and environment-friendly nanopesticide delivery systems are critical for the sustainable development of agriculture. In this study, a graphene oxide nanocomposite was developed for pesticide delivery and plant protection with pyraclostrobin as the model pesticide. First, graphene oxide-pyraclostrobin nanocomposite was prepared through fast adsorption of pyraclostrobin onto graphene oxide with a maximum loading of 87.04%. The as-prepared graphene oxide-pyraclostrobin nanocomposite exhibited high stability during two years of storage, suggesting its high potential in practical application. The graphene oxide-pyraclostrobin nanocomposite could achieve temperature (25 °C, 30 °C and 35 °C) and pH (5, 7 and 9) slow-release behavior, which overcomes the burst release of conventional pyraclostrobin formulation. Furthermore, graphene oxide-pyraclostrobin nanocomposite exhibited considerable antifungal activities against Fusarium graminearum and Sclerotinia sclerotiorum both in vitro and in vivo. The cotoxicity factor assay revealed that there was a synergistic interaction when graphene oxide and pyraclostrobin were combined at the ratio of 1:1 against the mycelial growth of Fusarium graminearum and Sclerotinia sclerotiorum with co-toxicity coefficient values exceeding 100 in vitro. The control efficacy of graphene oxide-pyraclostrobin nanocomposite was 71.35% and 62.32% against Fusarium graminearum and Sclerotinia sclerotiorum in greenhouse, respectively, which was higher than that of single graphene oxide and pyraclostrobin. In general, the present study provides a candidate nanoformulation for pathogenic fungal control in plants, and may also expand the application of graphene oxide materials in controlling plant fungal pathogens and sustainable agriculture.

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Extreme ecosystems are a possible source of new interesting microorganisms, in this study the isolation of psychrophilic and psychrotolerant plant growth promoting microorganisms was pursued in a cold habitat, with the aim of finding novel microbes that can protect crops from cold. Eight yeast and four bacterial strains were isolated from rhizospheric soil collected from the Xinantécatl volcano in Mexico, and characterized for plant growth promoting properties. Most of the yeasts produced indole acetic acid and hydrolytic enzymes (cellulases, xilanases and chitinases), but none of them produced siderophores, in contrast to their bacterial counterparts. Inorganic phosphate solubilization was detected for all the bacterial strains and for two yeast strains. Yeast and bacterial strains may inhibit growth of various pathogenic fungi, propounding a role in biological control. Microorganisms were identified up to genera level, by applying ribotyping techniques and phylogenetic analysis. Bacterial strains belonged to the genus Pseudomonas, whereas yeast strains consisted of Rhodotorula sp. (4), Mrakia sp. (3) and Naganishia sp. (1). New species belonging to the aforementioned genera seem to have been isolated from both bacteria and yeasts. Germination promoting activity on Solanum lycopersicum seeds was detected for all strains compared to a control, whereas tomato plantlets, grown at 15 °C in the presence of some of the strains, performed better than the non-inoculated plantlets. This study offers the possibility of using these strains as an additive to improve culture conditions of S. lycopersicum in a more environmentally compatible way. This is the first study to propose psychrophilic/psychrotolerant yeasts, as plant growth promoting microbes.

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Information on the sensitivity of spoilage fungi of bakery products to sanitizing agents is scarce in the literature. Thus, the aim of this study was to evaluate the antifungal activity of different classes of commercial sanitizers, which have permitted use in the food industry, on the main fungi involved in spoiling bakery products. The tests were carried out according to the protocol for testing the antifungal effect of chemical sanitizers of the European Committee for Standardization (CEN), with adaptations. Different strains of six isolated fungal species responsible for spoiling bakery products (Penicillium roqueforti, Penicillium paneum, Hyphopichia burtonii, and Aspergillus pseudoglaucus) were tested against five sanitizers at three concentrations: benzalkonium chloride (0.3%, 2.5%, 5%), biguanide (2%, 3.5%, 5%), peracetic acid (0.15%, 1.5%, 3%), quaternary ammonium (0.3%, 2.5%, 5%), and sodium hypochlorite (0.01%, 0.1%, 0.2%). Peracetic acid was the most effective sanitizes considering the genera, species, and concentrations evaluated, generally being capable of reductions between 2 and 4 logs of initial control tested. Biguanide should not be the compound of choice when the main goal of the bakery industry is fungal control.

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Nitric oxide (NO) is a major plant signaling molecule that plays key roles during plant-pathogen interactions and plant development. Previous work showed the participation of NO in the development and lignin composition of sunflower roots. Thereby, we have hypothesized that NO applications could control the attack of the fungal pathogen Verticillium dahliae in sunflowers. Seedlings growing hydroponically were pretreated with NO donors and further inoculated with the fungus. Evaluation of disease symptoms showed that NO pretreatments could not reduce Verticillium wilt. Strikingly, NO donors appear to promote the fungal infection. These results indicate that NO applications were unable to protect sunflowers from Verticillium attack and highlight the role played by the fine tuning regulation of NO levels required to balance plant responses between development and defense.

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