Effect of the Entomopathogenic Fungus Beauveria bassiana on the Development of Faba Bean (Vicia faba) Diseases in the Field Conditions.

Several ascomycetous entomopathogenic fungi, including species in the genera Beauveria, are plant symbionts/endophytes and are termed as endophytic insect-pathogenic fungi. It was shown that the fungus Beauveria bassiana (BBK-1 strain) successfully colonized Vicia faba bean plants in laboratory and field conditions of Western Siberia. The B. bassiana reisolate passed through the plants had significantly higher antagonistic activity against phytopathogens in comparison with the primary stem of entomopahogenic fungi. Pre-sowing faba bean seeds treatment reduced the level of infection of the seed material with phytopathogens, significantly decrease the development and prevalence of root rot disease. A decrease in the disease development index (chocolate spot, powdery mildew, fusariosis and other spots diseases) was found as a result of the use of B. bassiana. The effectiveness and prolonged action of B. bassiana on plants opens up new opportunities both in the creation of biological products and in molecular-genetic research and selection of certain pairs of plants and fungi based on the principle of the greatest synergy.

[COMPARATIVE ANALYSIS OF THE IMMUNE REACTIONS IN COLORADO POTATO BEETLE LARVAE UNDER DEVELOPMENT OF MYCOSES CAUSED BY METARHIZIUM ROBERTSII, M. BRUNNEUM AND M. PEMPHIGI].

A comparative investigation of humoral and cellular immune response in larvae of Colorado potato beetle Leptinotarsa decemlineata was conducted under development of mycoses caused by entomopatho- genic fungi Metarhizium robertsii, M. brunneum and M. pemphigi. The larvae were found highly suscep- tible to M. robertsii, M. brunneum and less susceptible to M. pemphigi. The susceptibility to the fungi was not correlated with the rate of conidia germination in epicuticular extracts of larvae. A non-specific for Colorado beetle pathogen M. pemphigi did not cause significant changes in the immune response and did not result in colonization of larvae hemocoel. Infection with M. robertsi and M. brunneum led to an increase in total hemocyte count at the initial stages of mycoses (day 2) followed by a sharp decrease on day 3. The strongest decrease was observed for the immunocompetent cells - plasmatocytes and granu- locytes. Enhanced phenoloxidase activity in hemolymph and cuticle was found on days 2 and 3 after in- fection. These changes in immune reactions correlated with the level of virulence of the strains. Thus, the immune response in Colorado potato beetle larvae is an important factor determining differences in the development of mycoses caused by different Metarhizium species.

Expression of amplified synthetic ethanol pathway integrated using Tn7-tool and powered at the expense of eliminated pta, ack, spo0A and spo0J during continuous syngas or CO2 /H2 blend fermentation.

AIMS: To engineer acetogen biocatalyst selectively overproducing ethanol from synthesis gas or CO2 /H2 as the only liquid carbonaceous product. METHODS AND RESULTS: Ethanol-resistant mutant originally capable of producing only acetate from CO2 /CO was engineered to eliminate acetate production and spore formation using our proprietary Cre-lox66/lox71-system. Bi-functional aldehyde/alcohol dehydrogenase was inserted into the chromosome of the engineered mutant using Tn7-based approach. Recombinants with three or six copies of the inserted gene produced 525 mmol l(-1) and 1018 mmol l(-1) of ethanol, respectively, in five independent single-step fermentation runs 25 days each (P < 0.005) in five independent repeats using syngas blend 60% CO and 40% H2 . Ethanol production was 64% if only CO2 + H2 blend was used compared with syngas blend (P < 0.005). CONCLUSIONS: Elimination of genes unnecessary for syngas fermentation can boost artificial integrated pathway performance. SIGNIFICANCE AND IMPACT OF THE STUDY: Cell energy released via elimination of phosphotransacetylase, acetate kinase and early-stage sporulation genes boosted ethanol production. Deletion of sporulation genes added theft-proof feature to the engineered biocatalyst. Production of ethanol from CO2 /H2 blend might be utilized as a tool to mitigate global warming proportional to CO2 fermentation scale.

Selective production of acetone during continuous synthesis gas fermentation by engineered biocatalyst Clostridium sp. MAceT113.

AIMS: To engineer acetogen biocatalyst capable of fermenting synthesis gas blend to acetone as the only liquid carbonaceous product. METHODS AND RESULTS: The metabolic engineering comprised inactivation of phosphotransacetylase via integration of a cassette comprising synthetic genes erm(B), thiolase and HMG-CoA synthase. Acetaldehyde dehydrogenase was inactivated via integration of a cassette consisting of synthetic genes cat, HMG-CoA lyase and acetoacetate decarboxylase. The engineered biocatalyst Clostridum sp. MAceT113 lost production of 253 mmol l(-1) ethanol and 296 mmol l(-1) acetate and started producing 1.8 mol l(-1) acetone in single-stage continuous syngas fermentation. CONCLUSIONS: The acetone concentration in culture broth is economical for bulk manufacture because it is about twenty times of that achieved with known acetone-butanol-ethanol fermentation of sugars. SIGNIFICANCE AND IMPACT OF THE STUDY: The process shows the opportunity to produce acetone from synthesis gas at concentrations comparable with production of acetone from products of petroleum cracking. This is the first report on elimination of acetate and acetaldehyde production and directing carbon flux from Acetyl-CoA to acetone via a non-naturally occurring in acetogen acetone biosynthesis pathway identified in eukaryotic organisms.

Electrotransformation of Clostridium acetobutylicum ATCC 824 using high-voltage radio frequency modulated square pulses.

Molecular biological improvement of industrial solventogenic clostridia could be enhanced by a higher efficiency of electrotransformation. In this research, we used a new approach to determine the frequency spontaneously generated by Clostridium acetobutylicum ATCC 824 cells during the application of a square high-voltage pulse. Once the frequency of 100 kHz was determined we transformed clostridial cells with pSOS84 plasmid DNA using radio-frequency modulated high-voltage square pulses (electric field strength 12 kVcm-1; pulse duration 22.5 ms; frequency of pulse modulation 100 kHz) to reach an efficiency exceeding 106 transformants microg-1 of plasmid DNA. We propose a possible role for cellular membrane structures in affecting the transformation yield.

Electrofusion of Escherichia coli cells.

Bacterial cell fusion was observed under conditions optimal for highly efficient electrotransformation of untreated Escherichia coli cells. E. coli clones possessing the joint phenotype were found after electric treatment of genetically marked parental strains. The phenotypes of the clones segregated due to a subsequent cultivation of the clones, whereas some of them turned out to be recombinants. Electron microscopy carried out 1 h after electric treatment of the cells revealed physical contacts between the cells being in parallel arrangement. The electrofusion processes detected are important for the estimation of electrotransformation efficiency. A possibility to use electrofusion of untreated bacterial cells for in vivo transfer of plasmid and chromosomal DNAs is discussed.