Volatile Organic Compound Profiles From Wheat Diseases Are Pathogen-Specific and Can Be Exploited for Disease Classification

Plants and fungi emit volatile organic compounds (VOCs) that are either constitutively produced or are produced in response to changes in their physico-chemical status. We hypothesized that these chemical signals could be utilized as diagnostic tools for plant diseases. VOCs from several common wheat pathogens in pure culture (Fusarium graminearum, Fusarium culmorum, Fusarium avenaceum, Fusarium poae, and Parastagonospora nodorum) were collected and compared among isolates of the same fungus, between pathogens from different species, and between pathogens causing different disease groups [Fusarium head blight (FHB) and Septoria nodorum blotch (SNB)]. In addition, we inoculated two wheat varieties with either F. graminearum or P. nodorum, while one variety was also inoculated with Blumeria graminis f.sp. tritici (powdery mildew, PM). VOCs were collected 7, 14, and 21 days after inoculation. Each fungal species in pure culture emitted a different VOC blend, and each isolate could be classified into its respective disease group based on VOCs with an accuracy of 71.4 and 84.2% for FHB and SNB, respectively. When all collection times were combined, the classification of the tested diseases was correct in 84 and 86% of all cases evaluated. Germacrene D and sativene, which were associated with FHB infection, and mellein and heptadecanone, which were associated with SNB infection, were consistently emitted by both wheat varieties. Wheat plants infected with PM emitted significant amounts of 1-octen-3-ol and 3,5,5-trimethyl-2-hexene. Our study suggests that VOC blends could be used to classify wheat diseases. This is the first step toward a real-time disease detection in the field based on chemical signatures of wheat diseases.

Comparative genomics reveals electron transfer and syntrophic mechanisms differentiating methanotrophic and methanogenic archaea

The anaerobic oxidation of methane coupled to sulfate reduction is a microbially mediated process requiring a syntrophic partnership between anaerobic methanotrophic (ANME) archaea and sulfate-reducing bacteria (SRB). Based on genome taxonomy, ANME lineages are polyphyletic within the phylum Halobacterota, none of which have been isolated in pure culture. Here, we reconstruct 28 ANME genomes from environmental metagenomes and flow sorted syntrophic consortia. Together with a reanalysis of previously published datasets, these genomes enable a comparative analysis of all marine ANME clades. We review the genomic features that separate ANME from their methanogenic relatives and identify what differentiates ANME clades. Large multiheme cytochromes and bioenergetic complexes predicted to be involved in novel electron bifurcation reactions are well distributed and conserved in the ANME archaea, while significant variations in the anabolic C1 pathways exists between clades. Our analysis raises the possibility that methylotrophic methanogenesis may have evolved from a methanotrophic ancestor.

CATTLE NECROBACTERIOSIS IN THE LIVESTOCK FACILITIES OF THE ALMATY REGION

Necrobacteriosis affects many species of animals. The most susceptible and sensitive to Fusobacterium necrophorum are reindeer, cattle and small cattle, pigs, and rabbits. A constant carriership of the causative agent of necrobacteriosis in the rumen and intestines of ruminants has been established, causative agent is found in food particles during chewing, as well as in feces. The causative agent of necrobacteriosis is widespread in the environment (livestock buildings, walking yards, manure, soil, pastures, stagnant reservoirs, etc.). Infestation of animals occurs when the pathogen enters the injured areas of the skin or mucous membranes of animals. Disturbed blood circulation, cracks and peeling of the horn happen as a result of long-term keeping of animals in damp premices, grazing them in damp, swampy areas, and also maceration of the limb tissues. Four cultures of the causative agent of cattle necrobacteriosis Fusobacterium necrophorum were isolated from sick animals with symptoms of lameness, their biological properties were studied. The pathogenicity of the isolated cultures was studied in laboratory animals. The work was conducted in laboratory and production conditions in "KazSRVI" LLP and at the dairy farm at "Arkabay" human settlement (village) of Talgar district of Almaty region, where stall keeping of animals is practiced. Slices from the diseased hoof of cows were taken at the border of the diseased and healthy tissue. Samples of the selected biological material were plated on Kitt-Tarozzi medium at the sampling site on the farm. The biological material taken from sick animals was studied within several hours after sampling in accordance with the guidelines for laboratory diagnosis of necrobacteriosis. Material for laboratory research (sections from the horny tissue of the hoof on the border with the healthy one) were taken fresh and inoculated on a nutrient medium for anaerobes. The results of cultivation of the necrobacteriosis causative agent on liquid and solid nutrient media under anaerobic conditions are presented. To get rid of the accompanying microflora and obtain a pure culture of F. necrophorum, a bioassay was set on laboratory animals - rabbits. All isolated cultures were highly pathogenic for rabbits. On the 14-15th day after infection, the experimental rabbits died. A pure culture of F. necrophorum, not contaminated with extraneous microflora, was sown from the internal organs of rabbits. It was found that rabbits are the optimal biomodel for purification of the F. necrophorum culture. The biochemical properties of the isolated cultures have been studied. It was found that epizootic cultures of the causative agent of necrobacteriosis emitted hydrogen sulfide and had hemolytic properties. In experiments in vitro and in vivo, it was found that the isolated cultures of F. necrophorum showed hyaluronidase activity. Cultures of F. necrophorum had a high catalase activity, they split hydrogen peroxide with the formation of oxygen (gas bubbles). When studying biochemical properties, it was found that F. necrophorum releases ammonia within 2-3 hours. Four cultures of F. necrophorum isolated from biological material from cattle were identical in biological properties.

Evaluation of the Antibacterial and Prebiotic Potential of Ascophyllum nodosum and Its Extracts Using Selected Bacterial Members of the Pig Gastrointestinal Microbiota

Ascophyllum nodosum and its extracts are promising antibacterial and prebiotic dietary supplements for pigs. The objectives of this study were to evaluate the effects of the increasing concentrations of: (1) two whole biomass samples of A. nodosum with different harvest seasons, February (ANWB-F) and November (ANWB-N), in a weaned pig faecal batch fermentation assay, and (2) A. nodosum extracts produced using four different extraction conditions of a hydrothermal-assisted extraction methodology (ANE1–4) and conventional extraction methods with water (ANWE) and ethanol (ANEE) as solvent in individual pure culture growth assays using a panel of beneficial and pathogenic bacterial strains. In the batch fermentation assay, ANWB-F reduced Bifidobacterium spp. counts (p < 0.05) while ANWB-N increased total bacterial counts and reduced Bifidobacterium spp. and Enterobacteriaceae counts (p < 0.05). Of the ANE1–4, produced from ANWB-F, ANWE and ANEE that were evaluated in the pure culture growth assays, the most interesting extracts were the ANE1 that reduced Salmonella Typhimurium, enterotoxigenic Escherichia coli and B. thermophilum counts and the ANE4 that stimulated B. thermophilum growth (p < 0.05). In conclusion, the extraction method and conditions influenced the bioactivities of the A. nodosum extracts with ANE1 and ANE4 exhibiting distinct antibacterial and prebiotic properties in vitro, respectively, that merit further exploration.

Development of Local Functional Food Made of Buffalo Milk Improved with Culture Probiotics (Lactobacillus casei)

The research was conducted to develop functional food products of milk-based livestock origin (Semi hard-type cheese), with the addition of pure culture Lactobacillus casei as a probiotic agent, and citric acid and Mucor meihei as milk coagulants. The research material was semi-hard type cheese made of approximately 35 liters of buffalo milk from West Sumbawa Regency as a basic ingredient with the probiotic pure culture. The results showed that the pure culture of probiotic (Lactobacillus casei) at levels of 10% and 15% can survive and develop quite well in semi-hard cheese during aging, from 1 day, 7 days, and 14 days, respectively (3.79 – 5.92) and (4.91 – 6.31) log cfu g-1. While the 0.025% rennet of the volume of milk from Mucor miehei gives a pretty good result, it can be seen from the product recovery which can reach (34.30 + 0.32) %. During aging for 14 days, an organoleptic quality which includes aroma, color, and texture was getting better, with the criteria of a semi-hard aroma, yellowish-white color, and semi-hard texture. It can be concluded, that semi-hard type cheese can be used as one of the functional foods of probiotic carriers. To get the therapeutic effect, this probiotic should be consumed at least 100 grams per serving.

The use of the strain Enterococcus faecium in the technology of yogurt “Carpathian” as an ancillary culture with probiotic properties

The results of research on the technological features of the production of yogurt "Carpathian" are covered in the article. The bacterial preparation of Chr. Hansen series YoFlex Premium 1.0 (L. bulgaricus, S. thermophilus) and Creamy 1.0 (L. bulgaricus, S. thermophilus, L. rhamnosus) and strain E. faecium SB 18, which is isolated from traditional Carpathian fermented products were used to produce research yogurt samples. It was found that when the strains were used together, the microorganisms were compatible, did not show interspecific antagonism and did not inhibit the enzymatic process. Based on yogurt microorganisms and E. faecium SB 18 strain, seven prototypes of yogurt were created: № 1 (100 %) – control, Premium + Creamy; № 2 (100 %) – control, pure culture of E. faecium SB 18; №3 (100:100 %) – control, (Premium + Creamy) + E. faecium SB 18; №4 (50:50 %) – (Premium + Creamy) + Ent. faecium SB 18; № 5 (70:30 %) – (Premium + Creamy) + Ent. faecium SB 18; № 6 (80:20 %) – (Premium + Creamy) + Ent. faecium SB 18; № 7 (90:10 %) – (Premium + Creamy) + Ent. faecium SB 18. The fastest fermentation took place in sample № 1 (pH 4.78 units – 4 h), the slowest in sample №2 (pH 4.81 units – 6 h), where only pure culture of E. faecium SB 18 was used. The fermentation time in sample №3 was initially slower and then more active (pH 4.77 units – 4 h). The acidity increased more moderately in samples № 4, 5, 6, 7 for 4 h, and at the end of fermentation was 4.84 units, 4.76 units, 4.81 units. and 4.75 units. in accordance. According to organoleptic evaluation, the experimental samples were characterized by slight differences. In general, it is noted that the addition of microbial culture of E. faecium SB 18 improves the taste of yogurt. Samples № 6 and № 7 with the addition of E. faecium SB 18 strain in the amount of 20 and 10 % were noted for the best organoleptic properties. According to the score scale, the above-mentioned samples received the highest number of points – 48, out of a possible 50. The dependence of the acidity of yogurt during storage was established on the dose and composition of the bacterial preparation. It was investigated that the acidity of yogurt, which contained an additional strain of E. faecium SB 18 in the ratios of 100:100 (sample 3) and 50:50 (sample 4), tends to increase rapidly in acidity, which is associated with increased lactic acid bacteria. It was found that partial replacement of the amount of traditional yogurt leaven in the ratio of 70:30 (sample 5), 80:20 (sample 6) and 90:10 (sample 7) provides the optimal course of the enzymatic process during fermentation and storage. It was found that the use of traditional yogurt leaven YoFlex Premium 1.0 and Creamy 1.0. together with strain E. faecium SB 18 in a ratio of 80:20, provides excellent consumer properties of the product.

Evaluation of the Effectiveness of the Fungicide Comissar Against Rice Pyricularia

The results of research on the effectiveness of the fungicide Comissar are presented, EC (active agents propiconazole, 300 g/l and tebuconazole, 200 g/l) against rice Pyricularia, in the conditions of laboratory and vegetation experiments. The experiment was conducted in 2016-2017 in the Primorsky Territory. The fungicide was used at the consumption rate of 0.3 l/ha and 0.4 l/ha one time. In the primary laboratory tests on pure culture Pyricularia oryzae a high inhibitory activity of the drug against the coastal population of rice Pyricularia was revealed. It is noticed that Comissar significantly inhibits the growth of fungal colonies in all tested concentrations. Treatment of the nutriculture medium with solutions of the drug provides a significant preventive effect on the development of P. oryzae compared to an untreated control. The effectiveness of the drug was 100% on the 7th day and 89.3-90.4% on the 14th day. It was found that the use of the drug as a fungicide for the treatment of vegetative rice plants significantly reduces the development of Pyricularia. Comissar has a high biological efficacy against the pathogen at 47.4% (0.3 l/ha) and 62.7% (0.4 l/ha). The use of the fungicide contributed to the active growth of plants and it increased the main productivity indicators: the length of the panicle by 2.08-32.17 cm, the number of filled grains by 3.0-4.5 pcs. and their mass by 0.21-0.24 g per plant, as well as the mass of 1000 grains by 4.91-5.51 g. The maximum reliable increase in grain yield by 3.77 g/vessel was obtained in the variant using a fungicide at the minimum consumption rate.

Proteomic Analysis of a Syntrophic Coculture of Syntrophobacter fumaroxidans MPOBT and Geobacter sulfurreducens PCAT

We established a syntrophic coculture of Syntrophobacter fumaroxidans MPOBT (SF) and Geobacter sulfurreducens PCAT (GS) growing on propionate and Fe(III). Neither of the bacteria was capable of growth on propionate and Fe(III) in pure culture. Propionate degradation by SF provides acetate, hydrogen, and/or formate that can be used as electron donors by GS with Fe(III) citrate as electron acceptor. Proteomic analyses of the SF-GS coculture revealed propionate conversion via the methylmalonyl-CoA (MMC) pathway by SF. The possibility of interspecies electron transfer (IET) via direct (DIET) and/or hydrogen/formate transfer (HFIT) was investigated by comparing the differential abundance of associated proteins in SF-GS coculture against (i) SF coculture with Methanospirillum hungatei (SF-MH), which relies on HFIT, (ii) GS pure culture growing on acetate, formate, hydrogen as propionate products, and Fe(III). We noted some evidence for DIET in the SF-GS coculture, i.e., GS in the coculture showed significantly lower abundance of uptake hydrogenase (43-fold) and formate dehydrogenase (45-fold) and significantly higher abundance of proteins related to acetate metabolism (i.e., GltA; 62-fold) compared to GS pure culture. Moreover, SF in the SF-GS coculture showed significantly lower abundance of IET-related formate dehydrogenases, Fdh3 (51-fold) and Fdh5 (29-fold), and the rate of propionate conversion in SF-GS was 8-fold lower than in the SF-MH coculture. In contrast, compared to GS pure culture, we found lower abundance of pilus-associated cytochrome OmcS (2-fold) and piliA (5-fold) in the SF-GS coculture that is suggested to be necessary for DIET. Furthermore, neither visible aggregates formed in the SF-GS coculture, nor the pili-E of SF (suggested as e-pili) were detected. These findings suggest that the IET mechanism is complex in the SF-GS coculture and can be mediated by several mechanisms rather than one discrete pathway. Our study can be further useful in understanding syntrophic propionate degradation in bioelectrochemical and anaerobic digestion systems.

THE USE OF NANO AND BIOTECHNOLOGICAL RAW MATERIALS IN THE CREATION OF DRUGS FOR AGRICULTURE

The article is devoted to the consideration of the possibility of using fullerene-containing water as part of humic growth-stimulating preparations for agriculture with the addition of yeast production waste. More recently, the problems of the economy and the environment were perceived as opposite. Currently, there is a need for a mutually dependent and mutually beneficial combination of economic and environmental interests, which was the basis for this study. The analysis of the wastewater of the yeast-producing enterprise shows the presence of a large number of chemical and organic substances that pose a certain danger to the environment, but are useful after processing waste for use in agriculture. The largest number of substances is found in waste, after the stage of production of pure culture. The experiment shows that the use of structured water in combination with biologically active waste from the production of bread yeast obtained on the basis of the Saccharomyces cerevisiae strain increases the yield of grain crops. Based on the established fact of a wide and universal spectrum of biological activity of the water-carbon structure of hydrated fullerenes, according to patent and scientific and technical literature, they have not been widely tested as plant growth regulators. We will investigate the interaction of substances with water structured with fullerenes, the effect of surfactants on the formation and stability of emulsified humates with film-forming agents, the adhesive properties of the components, the effect of concentrated yeast fermentation liquid separated after the stage of growing a pure culture of baking yeast, the effect of both a fullerene-like structure – shungite of natural origin and hydrated fullerenes – highly stable finely dispersed aqueous solutions of native fullerenes (have the properties of lyophobic molecular colloidal systems) on grain yield, protection of vegetative plants. The result of the work will be the substantiation of the principles of the methodology for the application of nanostructured substances for use in biotechnologies of multifunctional highly effective drugs for agriculture with biologically active additives.

Watershed: A Key for Microbial Biogeography

Biogeography research is flawed by the poor understanding of microbial distributions due to the lack of a systematic research framework, especially regarding appropriate study units. By combining pure culture and molecular methods, we studied the biogeographic patterns of nematode-trapping fungi by collecting and analysing 2,250 specimens from 228 sites in Yunnan Province, China. We found typical watershed patterns at the species and genetic levels of nematode-trapping fungi. The results showed that microbial biogeography could be better understood by 1) using watersheds as research units, 2) removing the coverup of widespread species, and 3) applying good sampling efforts and strategies. We suggest that watersheds could help unify the understanding of the biogeographic patterns of animals, plants, and microbes and may also help account for the historical and contemporary factors driving species distributions.