ANTIMICROBIAL, ENTOMOPATHOGENIC AND ANTIVIRAL ACTIVITY OF GAUPSIN BIOPREPARATION CREATED ON THE BASIS OF Pseudomonas chlororaphis STRAINS

subsp. aureofaciens, antibiotics – phenazines, pyrrolnitrine, entomopathogenic properties, antiviral activity

The aim of this review was to present the results of more than ten-year study of gaupsin biopreparation created on the basis of two strains Pseudomonas chlororaphis subsp.aureofaciens UCM В-111 and UCM В-306 with antifungal, entomopathogenic and antiviral activities.Data about antibiotic substances produced by these strains -phenazine and phenylpyrrole derivativesare presented.Entomocidal properties against the wide spectrum of insect pests have been found out in the strains-producers.Antiviral activity of gaupsin due to the production of thermostable exopolymers containing neutral monosaccharides has been shown using the tobacco mosaic virus as a model.Lipopolysaccharides of the strains В-111 and В-306 also appeared to be highly active antiviral agents.Structure of their O-specific polysaccharides has been established.The last one are structurally heterogenic, presented by linear tri-and tetrasaccharide repeated links and have specific structure that has not been described previously.

REVIEWS
BIOTECHNOLOGIA ACTA, V. 10, No 1, 2017 strains with a culture medium.In recent years, we have established some new properties of strains that are the gaupsin components, expanding the understanding of their biological activity and the prospects of their use in the pathogens and plant pests control.
Below we'll mention briefly some important features of the biological activity of P. chlororaphis subsp.aureofaciens strainscomponents of the biopreparation gaupsin and the substances produced by them and determining its activity.Some of these substances are found in many strains of the genus Pseudomonas and have been studied in detail; others have been discovered and studied by us for the first time.

Antimicrobial substances of strainscomponents of the preparation gaupsin
Up to the present, phytopathogenic fungi and, to a lesser extent, bacteria, are considered as the main target of the P. chlororaphis strains action.Siderophores -substances that carry out the iron transport, especially pseudobactin (pyoverdin) -the yellow-green fluorescent pigment of bacteria of the genus Pseudomonas, belong to the compounds that play an important role in limiting the number of pathogens [8].Pyoverdin is the instrument of bacteria competition for iron with siderophores of phytopathogenic fungi, which have a lower iron binding constant.Thus, the fungistatic effect of pseudobactin is associated with the creation of iron deficiency for fungi -plant pathogens.Strains B-111 and B-306, which are the gaupsin components, actively synthesize pseudobactin, as evidenced by yellow-green fluorescence at their cultivation in both commonly used and special media.
Chromato-mass spectrometric studies have shown the presence of phenazine derivatives both directly in gaupsin (on a production medium) and when growing of bacteria on King A medium, which is optimal for this group of pigments biosynthesis.
For a long time phenazines were considered only as antimicrobial compounds.However, in the last decades these ideas have been revised and supplemented by numerous experimental data demonstrating the important role of phenazines in the microbial cell: as electron carriers possessing a significant redox potential and affecting the reduction of Fe 3+ to Fe 2+ ions that are more accessible for both bacteria and plants, as signaling molecules that induce through the expression of genes the mechanisms of plant protection from infection, as well as agents that play an important role in the formation of microbial biofilm, and many other properties [1,2,10].The formation of biofilms on the roots of plants with fluorescent bacteria of the genus Pseudomonas and the participation of phenazines in these processes has been the subject of numerous reports.It has been found that phenazine-1-carboxylic acid affects the growth of cells in the biofilm itself, and 2-hydroxyphenazine-1-carboxylic acid, synthesized by the strain P. chlororaphis, simultaneously with the above-mentioned compound affects on the adhesion of cells to surfaces [11].All these properties along with antibiotic activity determine the use of pseudomonads for biological protection of plants and their productivity increase.
With the ability to synthesize antibiotics of the phenazine group (and possibly different chemical nature compounds as well), the action of P. chlororaphis strains on phytopathogenic nematodes, which inhabit the soil extensively and cause significant crop losses of up to 70%, is associated [12].In our experiments, the strains of this species were the most active antagonists of the stem nematode of potato Ditylenchus destructor in comparison with other species of pseudomonads [13].Pyrrolnitrine contributes to the antifungal effect of gaupsin; it is active antifungal agent of the phenylpyrrole group.We detected this antibiotic in a culture liquid of the strain B-306.It is effective against Gram-positive and Gram-negative bacteria and acts on many species of parasitic and saprophytic fungi in concentration of 0.19-10 μg/ml.This antibiotic was not detected in the culture liquid of the strain B-111.
Thus, the compounds responsible for the antifungal and antibacterial activity of the preparation gaupsin are, according to our data, derivatives of phenazine and phenylpyrrole.The intensity of the biosynthesis of these antibiotics by the bacteria that are the part of the preparation and their activity against fungi and bacteria -causative agents of plant diseases is characterized.

Effect of strains-components of the preparation gaupsin on insect-pests
No less important than the antimicrobial, but much less studied is the entomopathogenic activity of P. chlororaphis strains.Ten supported in the UCM (Ukrainian Collection of Microorganisms) strains of P. chlororaphis subsp.aureofaciens limited the number of apple moths caterpillars of the 5 th age (mortality 26-85%) and the Colorado potato beetle larvae of the 4 th age (mortality 25-60%).In this case, insects perished from septicemia and the action of an entomopathogenic toxin [7].The most active strain was the UCM B-306; based on these experiments it was included into the composition of gaupsin.Many other insectpests are sensitive to gaupsin (Table 2, 3).
For many decades, the point of view (which also was reflected in the diagnosis of the genus Pseudomonas) was generally accepted, that pseudomonads do not possess entomopathogenic activity.However, in 2006 a species of P. entomophila, highly pathogenic for many insects belonging to different orders, was described [14].A complete sequencing of the genome of a type strain of the named species was carried out and the presence of an insecticidal toxin with high haemolytic activity, lipases and extracellular proteases involved in the death of insects was made in these bacteria.In 2012, by genetic analysis methods with the full sequencing of the genome of the Pseudomonas chlororaphis GP72 strain, used for biocontrol and colonizing the root system of plants, the Chinese authors identified an insecticidal toxin gene [15].Note: "-" -the activity against the test culture was not investigated.According to our data, some of the enzymes mentioned above are also present in strains of P. chlororaphis subsp.aureofaciens [13].However, the nature of the toxin formed by representatives of this species has not yet been established and requires study.

Antiviral activity of the preparation gaupsin
In 2010, studies were begun on the antiviral activity of gaupsin and the compounds responsible for it.It has been established that during three growth seasons gaupsin inhibited the development of the tobacco mosaic virus (TMV) of the strain U1 in experiments in vivo in Datura stramonium and Nicotiana tabacum cultures for 80-97% [9,16,17].Active with respect to TMV were culture fluids obtained both with joint (gaupsin) and with separate growth of the strains B-111 and B-306 on the production medium, and on the medium King A as well.
Water-soluble thermostable preparations were obtained from the fermentation medium by evaporation, dialysis and lyophilization.Both dialyzed and nondialyzed preparations exhibited similar antiviral activity, which indicated the high molecular weight of antiviral agents.The most significant effect (a decrease in the infectivity of TMV by 76.0-97.5%) on the leaves of tobacco and stramonium was obtained at a concentration of 10 mg/ml; the antiviral effect progressively decreased at concentrations of 1.0-0.1 mg/ml (40-57 and 24-27%, respectively).
The antiviral substances of gaupsin were not inactivated at 100 C for 10 min and, apparently, do not contain protein components.The results of the analysis indicate that they are exopolymers containing neutral monosaccharides (fucose, mannose, galactose and glucose).Their chemical structure is not established.
Highly active antiviral agents were lipopolysaccharides (LPS), isolated from cells of the strains B-111 and B-306 [18][19][20][21][22][23].They suppressed the infectivity of TMV on the model of three species of indicator plants (stramonium Datura stramonium L., tobacco Nicotiana tabacum L., variety Immune 580, and Nicotiana sanderae H.) at 0.001-10.0mg/ml.The O-specific side chains of LPS of both strains did not inhibit TMV, and often stimulated the development of the virus.At the same time, core oligosaccharides inhibited the development of viral infection to varying degrees.
It is known that lipopolysaccharides of growth-stimulating rhizobacteria can act as signaling molecules, causing nonspecific activation of plant protective mechanisms (induced systemic resistance -ISR) [19].It can be assumed that this mechanism may cause the antiviral effect of lipopolysaccharides of the strains -gaupsin components.At the same time, according to electron microscopy data, obtained during direct contact of LPS with the virus in vitro, the virions "stick together" forming "ligaments", while in the control separate free virus particles were observed, which indicates a direct interaction between the lipopolysaccharides of the strains of P. chlororaphis subsp.aureofaciens and TMV.
Thus, for the first time, the antiphytoviral effect of the gaupsin preparation and the strains of P. chlororaphis subsp.aureofaciens in its composition has been found.It is shown that high antiviral activity is caused both by the synthesis of exopolysaccharides and by the lipopolysaccharides of the strainsgaupsin components, and in the latter one by core oligosaccharides.Further studies of P. chlororaphis subsp.aureofaciens LPS have demonstrated their high effect in respect of viruses of warm-blooded -influenza, herpes and hepatitis C [24].
The next stage of the work, carried out in 2015-2016, was to clarify the O-specific polysaccharides structure of the strains B-306 and B-111, as well as of the P. chlororaphis UCM B-106 type strain [21,22].
It is shown that O-polysaccharides are structurally heterogeneous, represented by linear repeating tri-and tetrasaccharide units and possess a previously undescribed unique structure, and probably contribute to the high biological activity of P. chlororaphis lipopolysaccharides.

Gaupsin practical application
According to our data, the biopreparation gaupsin, as well as the strains of P. chlororaphis subsp.aureofaciens, has a wide range of properties that provide effective protection of agricultural plants.First of all, it is the synthesis of antifungal and antibacterial substances that inhibit the causative agents of plant diseases, which are thoroughly studied and widely used in the world practice of biological control, and also much less studied entomopathogenic properties (the nature of the insecticidal toxin has not been studied) and, finally, antiviral activity not known previously.The nature and (partly) the structure of substances that inhibit phytoviruses have been established, but the role of these compounds as biocontrol instruments has to be evaluated.
Gaupsin activity is directed against a wide range of pathogens.Most of them are also a target of the action of chemical plant protection products used in agriculture.All studied strains of P. chlororaphis were resistant to modern chemical fungicides and insect growth regulators [25].The resistance of bacteria to these herbicides suggests the possibility of their joint use, which could lead to a net beneficial effect, as well as the reduction in chemical preparations consumption, and significant reduction of the pesticide press to the biocenosis, and the production of output yield free from harmful chemicals.
At present, the biopreparation gaupsin is produced by a number of Ukrainian enterprises in accordance with the licensing agreements for release and sale, concluded with the developer of the gaupsin -Zabolotny Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine.Our partners -SE Enzyme, Ltd. "Research and Production Center Cherkasy Bio Protection", Ltd. "Company BioNik", Ltd. "Agricultural biotechnology" -established the production of the preparation, the main consumers of which are small and medium-sized farms, as well as private individuals.
Since the release of the preparation on the agricultural market up to the present day, numerous data have been obtained, confirming the high effectiveness of gaupsin as a polyfunctional biological agent with pronounced fungicidal, insecticidal and growth-stimulating activity.Below we will confirm this conclusion with several examples.Thus, Table 4 illustrates the fungicidal effect of the preparation for the protection of soybean from a number of the most common fungal diseases in western Polissya (2014).Soybean treatment with gaupsin (5 l/ha) inhibited the development of diseases at a level similar to, or slightly below, the Abacus fungicide (BASF).This chemical preparation is a two-component fungicide of the new generation, containing epoxiconazole and pyraclostrobin.It is used to control dangerous diseases of cereals, corn, soybeans and sugar beet and presented in the Ukrainian market as a premium fungicide for soybean use.
Gaupsin also shows a high activity for the protection of plants from insect pests: codling moth, aphids, pyralid moths (snout moths, grass moths, bud moth or flyweed), bugs, cicadas, moths, Colorado beetle, Owlet moths, wheat thrips, ground beetles, geometer moths, pyralid moths (snout moths or grass moths), mites.The effectiveness of gaupsin applying to protect apple trees from red spider mites (8 l/ha + EPPA-10 0.25 l/ha) was shown by production researches conducted in 2013 at the Ltd. "Agro firm Bilozerske" of the Kherson region (Table 5).
As a control, a tank mixture of chemical preparations Omite (2 l/ha) and Apollo (0.4 l/ha) was chosen.The active ingredients of the aforementioned acaricides of contact action are propargite and clofentezine, respectively, which are evaluated as highly dangerous and moderately dangerous substances for humans.The efficiency of a tank mixture of chemical preparations was 84.9 % compared to 82.7 % after treatment with gaupsin.Subsequent treatments of apple trees with chemical preparations increased the rate of insecticide application and led to an increase in pest resistance.

Pseudomonas spp. and biopraparat Gaupsin
Reviews 13 At the same time, the cost of chemical treatment was 3 times higher than of biological treatment.
Thus, a wide spectrum of biological activity favorably distinguishes gaupsin from numerous monovalent preparations directed against one (or one group) of plant pests.Current issues remain related to the substances responsible for the beneficial effect of gaupsin: the elucidation of the chemical structure of exopolymers responsible for the antiviral effect of the preparation; the study of the mechanisms of its action and effectiveness in field experiments against economically important pathogens of viral diseases of agricultural plants in Ukraine; the elucidation of the nature of the entomopathogenic toxin formed by bacteria; the further improvement of biotechnology and various forms of gaupsin usage.