Selection of Antarctic yeasts as gray mold biocontrol agents in strawberry.

The postharvest disease popularly known as gray mold is considered one of the most limiting factors strawberry fruit production. The most effective way to control this disease is still the use of chemical fungicides. However, other alternative sources of control are being explored. Among these, psychrophilic yeasts adapted to extreme conditions, such as those found in the Antarctic region, may have great potential for use as biocontrol agents. Thus, the present study aimed to select psychrotolerant yeasts obtained from Antarctic region and to evaluate their potential for biocontrol under gray mold, caused by Botrytis cinerea in strawberries stored at low temperature. For this, 20 potential antagonist yeasts were evaluated in vitro (thermotolerance and enzymatic) assays. Debaryomyces hansenii, Rhodotorula mucilaginosa and Dioszegia hungarica were selected for growing in strawberry juice. However, only D. hansenii was selected for in vivo studies and showed a reduction in the incidence of gray mold by 82% for the tests performed on injury and 86% for the tests on non-injured fruits treated by immersion bath. Thus, demonstrating that the selection of this cold-adapted Antarctic yeast can be a promising strategy as a biocontrol agent used to curb the development of gray mold in strawberry fruits.

Effect of alternative fungicides and inoculation strategy on yeast biodiversity and dynamics from the vineyard to the winery.

Fungi and oomycetes found in vineyards cause diseases such as powdery and downy mildew. Consequently, conventional and alternative agronomical practices are widely used prior to harvest to protect grapes. Alternative products are considered more eco-friendly and environmentally sustainable in comparison to conventional chemical products. However, the effect of these alternative products on yeast ecology, from the vineyard to the winery, is poorly understood. This study compared the effect of alternative and conventional chemical antifungal compounds (copper and sulphur based) on grapes' mycobiota in the vineyard and during subsequent winery fermentation using culture-dependent and -independent approaches. Culture-dependent data indicated a treatment-dependent effect on the load and diversity of yeast populations on grapes. It was found that the population of Hanseniaspora uvarum was higher on grapes previously treated with laminarin and copper, compared to the other levels registered on grapes previously treated with the rest of antifungal products tested in this study (including the untreated and conventional treatment controls). Concerning, wine quality, the chemical composition was not correlated to the application of antifungal treatment in the vineyard. Understanding the effect of different antifungal products on grape and wine microbial communities may help in setting up guidelines for wine grape production. These guidelines, can be used to guarantee quality in the pursuit of a sustainable competitive advantage in the market.

Selection and application of antifungal VOCs-producing yeasts as biocontrol agents of grey mould in fruits.

Rotting caused by grey mould (Botrytis cinerea) is a concerning disease for numerous crops both pre- and postharvest stages. Application of antagonistic yeasts is a promising strategy for controlling grey mould incidence which could mitigate undesirable consequences of using synthetic fungicides. In this work, a screening for detection of yeasts isolated from figs producers of antifungal volatile organic compounds (VOCs) were performed by confrontation in double dishes systems. Eleven out of 34 yeasts confronted reduced B. cinerea growth parameter in vitro. This reduction was correlated (p ≤ 0.050) with the production of 10 volatile compounds: two acids (acetic acid and octanoic acid), 7 esters (Ethyl propionate, n-Propyl acetate, Isobutyl acetate, 2-methylbutyl acetate, furfuryl acetate, phenylmethyl acetate, 2-phenylethyl acetate) and one ketone (Heptan-2-one). In bases on in vitro assay, Hanseniaspora uvarum 793 was applied to in vivo assays with strawberries and cherries. The reduction of incidence of B. cinerea in strawberries at 7 °C and 25 °C was 54.9 and 72.1% after 6 and 3 days, respectively. The reduction of incidence of B. cinerea in cherries at 7 °C and 25 °C was 48.9 and 45.6% after 5 and 4 days, respectively. These results showed that VOCs produced by Hanseniaspora uvarum 793 are effective in the control of incidence of Botrytis cinerea in fruits, being a potential alternative to chemical fungicide.

Agrochemicals: Effect on genetic resistance in yeasts colonizing winter wheat kernels.

Crop protection agents are widely used in modern agriculture and exert direct effects on non-target microorganisms such as yeasts. Yeasts abundantly colonize wheat grain and affect its chemical composition. They can also limit pathogen growth. This study evaluated the sensitivity of yeast communities colonizing winter wheat kernels to benzimidazole, strobilurin, triazole and morpholine fungicides, trinexapac-ethyl, a commercial mixture of o-nitrophenol+p-nitrophenol+5-nitroguaiacol, and chitosan applied during the growing season of winter wheat and in vitro in a diffusion test. A molecular identification analysis of yeasts isolated from winter wheat kernels was performed, and nucleotide polymorphisms in the CYTb gene (G143A) conferring resistance to strobilurin fungicides in yeast cells were identified. The size of yeast communities increased during grain storage, and the total counts of endophytic yeasts were significantly (85%) reduced following intensive fungicide treatment (fenpropimorph, a commercial mixture of pyraclostrobin, epoxiconazole and thiophanate-methyl). This study demonstrated that agrochemical residues in wheat grain can drive selection of yeast communities for reduced sensitivity to xenobiotics. A mutation in the CYTb gene (G143A) was observed in all analyzed isolates of the following azoxystrobin-resistant species: Aureobasidium pullulans, Debaryomyces hansenii, Candida albicans and C. sake. Agrochemicals tested in vitro were divided into four classes of toxicity to yeasts: (1) tebuconazole and a commercial mixture of flusilazole and carbendazim - most toxic to yeasts; (2) fenpropimorph and a commercial mixture of pyraclostrobin and epoxyconazole; (3) propiconazole, chitosan, thiophanate-methyl and a commercial mixture of o-nitrophenol, p-nitrophenol and 5-nitroguaiacol; (4) trinexapac-ethyl and azoxystrobin - least toxic to yeasts. It was found that agrochemicals can have an adverse effect on yeast abundance and the composition of yeast communities, mostly due to differences in fungicide resistance between yeast species, including the clinically significant C. albicans.

Differential role of HAMP-like linkers in regulating the functionality of the group III histidine kinase DhNik1p.

Nik1 orthologs are sensor kinases that function upstream of the high osmolarity glycerol/p38 MAPK pathway in fungi. They contain a poly-HAMP module at their N terminus, which plays a pivotal role in osmosensing as well as fungal death upon exposure to fludioxonil. DhNik1p is a typical member of this class that contains five HAMP domains and four HAMP-like linkers. We investigated the contribution of each of the HAMP-like linker regions to the functionality of DhNik1p and found that the HAMP4b linker was essential as its deletion resulted in the complete loss of activity. Replacement of this linker with flexible peptide sequences did not restore DhNik1p activity. Thus, the HAMP-like sequence and possibly structural features of this linker region are indispensable for the kinase activity of DhNik1p. To gain insight into the global shape of the poly-HAMP module in DhNik1p (HAMP1­5), multi-angle laser light and small angle x-ray scattering studies were carried out. Those data demonstrate that the maltose-binding protein-tagged HAMP1­5 protein exist as a dimer in solution with an elongated shape of maximum linear dimension ∼365 Å. Placement of a sequence similarity based model of the HAMP1­5 protein inside experimental data-based models showed how two chains of HAMP1­5 are entwined on each other and the overall structure retained a periodicity. Normal mode analysis of the structural model is consistent with the H4b linker being a key to native-like collective motion in the protein. Overall, our shape-function studies reveal how different elements in the HAMP1­5 structure mediate its function.

Inhibitory activity against plant pathogenic fungi of extracts from Myoporum bontioides A. Gray and indentification of active ingredients.

BACKGROUND: In order to understand the bioactivity of Myoporum bontioides A. Gray against plant pathogens and determine its active ingredients, the inhibitory activities of methanol extracts from M. bontioides against Fusarium oxysporum f. sp. niveum (E. F. Smith) Snyder & Hansen, Pestalotia mangiferae P. Henn., Thielaviopsis paradoxa (De Seynes) v. Hohnel, Colletotrichum musae (Berk. & M. A. Curtis) v. Arx, Alternaria alternata (Fr.) Keissler, Mycosphaerella sentina (Fr.) Schroter and Sphaceloma fawcettii Jenk. were evaluated using a growth rate method, and the active ingredient was isolated by activity-directed isolation and identified by determination and analysis of IR, (1)H NMR, (13)C NMR and mass spectra and correlative physical constants. RESULTS: The results showed that the extracts from stems and leaves of M. bontioides exhibited inhibitory activity against the seven fungi, with > 58% inhibition at 10 g L(-1) after 72 h. The active compound was isolated and identified as (-)-epingaione, and showed inhibitory activity against the above seven fungi. The inhibitory activity against P. mangiferae was the highest, with an EC(50) value of 77 mg L(-1). The EC(50) values against the other six fungi were 147-245 mg L(-1). (-)-Epingaione also inhibited spore germination of F. oxysporum f. sp. niveum, T. paradoxa and S. fawcettii. CONCLUSION: (-)-Epingaione demonstrated broad-spectrum inhibitory activity against plant pathogenic fungi and is promising for exploitation as a fungicide.

Cytochrome b gene structure and consequences for resistance to Qo inhibitor fungicides in plant pathogens.

The cytochrome b (cyt b) gene structure was characterized for different agronomically important plant pathogens, such as Puccinia recondita f sp tritici (Erikss) CO Johnston, P graminis f sp tritici Erikss and Hennings, P striiformis f sp tritici Erikss, P coronata f sp avenae P Syd & Syd, P hordei GH Otth, P recondita f sp secalis Roberge, P sorghi Schwein, P horiana Henn, Uromyces appendiculatus (Pers) Unger, Phakopsora pachyrhizi Syd & P Syd, Hemileia vastatrix Berk & Broome, Alternaria solani Sorauer, A alternata (Fr) Keissl and Plasmopara viticola (Berk & Curt) Berlese & de Toni. The sequenced fragment included the two hot spot regions in which mutations conferring resistance to QoI fungicides may occur. The cyt b gene structure of these pathogens was compared with that of other species from public databases, including the strobilurin-producing fungus Mycena galopoda (Pers) P Kumm, Saccharomyces cerevisiae Meyer ex Hansen, Venturia inaequalis (Cooke) Winter and Mycosphaerella fijiensis Morelet. In all rust species, as well as in A solani, resistance to QoI fungicides caused by the mutation G143A has never been reported. A type I intron was observed directly after the codon for glycine at position 143 in these species. This intron was absent in pathogens such as A alternata, Blumeria graminis (DC) Speer, Pyricularia grisea Sacc, Mycosphaerella graminicola (Fuckel) J Schröt, M fijiensis, V inaequalis and P viticola, in which resistance to QoI fungicides has occurred and the glycine is replaced by alanine at position 143 in the resistant genotype. The present authors predict that a nucleotide substitution in codon 143 would prevent splicing of the intron, leading to a deficient cytochrome b, which is lethal. As a consequence, the evolution of resistance to QoI fungicides based on G143A is not likely to evolve in pathogens carrying an intron directly after this codon.