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UDC 579.841.1:57.063.8:632.95 https://doi.org/10.15407/biotech10.01.007
ANTIMICROBIAL, ENTOMOPATHOGENIC AND ANTIVIRAL ACTIVITY OF GAUPSIN BIOPREPARATION CREATED ON THE BASIS OF Pseudomonas chlororaphis STRAINS
E. A. KIPRIANOVA1, L. A. SAFRONOVA1, A. O. PROSYANOV2
1Zabolotny Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine, Kyiv 2Ltd. Company "BioNik", Mykolaiv
E-mail: safronova_larisa@ukr.net
Received 29.12.2016
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 B-111 and UCM B-306 with antifungal, entomopathogenic and antiviral activities. Data about antibiotic substances produced by these strains — phenazine and phenylpyrrole derivatives — are 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 B-111 and B-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.
Key words: gaupsin biopreparation, Pseudomonas chlororaphis subsp. aureofaciens, antibiotics -phenazines, pyrrolnitrine, entomopathogenic properties, antiviral activity.
Saprophytic bacteria Pseudomonas chlororaphis belong to one of the most active producers of antibiotic substances among various species of pseudomonads [1, 2]. In recent decades, significant progress has been made in their use for the biological protection of plants from pathogens [3, 4]. Preparations containing strains of this species are widely used throughout the world for plants protection of fungal diseases. It is enough to refer to the strain Pseudomonas chlororaphis MA 342, isolated in Sweden in 1991, offered to protect cereals of these diseases. Biopreparation based on this strain was patented by the Swedish company Bio Agri AB under the trade name Cedomon, registered and used in many European countries [5]. According to the US Environmental Protection Agency conclusion, P. chlororaphis strains are
non- pathogenic and non-toxic to humans, biota and environment [6].
At the Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine, the complex biopreparation gaupsin based on two strains of P. chlororaphis subsp. aureofaciens, which inhibits the growth of phytopathogenic bacteria and fungi, was created and patented in Ukraine in 2005 [7]. The strains of P. chlororaphis subsp. aureofaciens UCM B-111 and UCM B-306 (UCM — Ukrainian collection of microorganisms) in gaupsin composition were selected in a large-scale study of the antagonistic activity of a large collection of bacteria of the genus Pseudomonas against phytopathogenic fungi and bacteria, as well as study of their effect on some pests. The preparation is a cellular biomass of both
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 strains — components 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 strains-components 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.
The antibiotics of phenazine group are important representatives of antimicrobial compounds formed by P. chlororaphis [1, 2, 9, 10], studied in detail in three species of pseudomonads: soil isolates of P. fluorescens and P. chlororaphis and opportunistic human pathogen P. aeruginosa. Three phenazine pigments were isolated from the strains-components of the biopreparation gaupsin: yellow phenazine-1-carboxylic acid, orange 2-hydroxyphenazine-1-carboxylic acid and 2-hydroxyphenazine.
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.
At the same time, the B-111 strain possessed a high phenazine-synthesizing activity, 520-600 mg/l of phenazine-1-carboxylic acid, 90-128 mg/l of 2-hydro-xyphenazine-1-carboxylic acid and 20-35 mg of 2-oxyphenazine were present in the culture liquid. The same compounds were present in the culture liquid of strain B-306, but in smaller amounts (350-380, 60-75 and 15-20 mg/l, respectively).
Table 1 presents the results of studying the antifungal activity of phenazine pigments synthesized by strains B-111 and B-306. All of them inhibit the growth of phytopathogenic fungi (2-hydroxyphenazine-1-carboxylic acid is the most active among them). These substances inhibit also the growth of bacteria-plant pathogens: Clavibacter michiganense, Agrobacterium tumefaciens, and Xantho-monas malvacearum. Phenazine-1-carboxylic acid acts on them in concentrations of 100-200 pg/ml; 2-hydroxyphenazine-1-car-boxylic acid — of 5-50 pg/ml, 2-hydroxy-phenazine — of 50-20 pg/ml, respectively.
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 Fe3+ to Fe2+ 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
Table 1. Antifungal activity of phenazine pigments synthesized by Pseudomonas chlororaphis subsp. aureofaciens strains — gaupsin components [9]
Phytopathogenic fungi Minimal fungi growth inhibitory concentration, ^g/ml
Phenazine-1-carboxylic acid 2-Hydroxyphenazine-1-carboxylic acid 2-Hydroxyphenazine
Fusarium avenaceum 200 200 400
Fusarium oxysporum 200 200 -
Mucor plumbeus 200 100 200
Drechslera graminea 50 20 50
Rhizopus arrhizus 100 100 -
Rhizoctonia solani 200 200 -
Monilia fructigena 200 100 200
Alternaria sp. 100 50 -
Botrytis cynerea 50 20 100
Note: "-" — the activity against the test culture was not investigated.
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 pg/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 5th age (mortality 26-85%) and the Colorado potato beetle larvae of the 4th 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 insect-pests 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].
Table 2. Entomocide activity of Pseudomonas chlororaphis subsp. aureofaciens strains against caterpillars
of apple moth and larvae of the Colorado potato beetle [7]
Strains P. chlororaphis subsp. aureofaciens Apple moth caterpillars (L5) Colorado potato beetle larvae (L4)
Total mortality,% Imago yield,% Total mortality,% Imago yield,%
B-108 31 69 45 55
B-109 80** 20 30 70
B-110 30 70 30 70
B-111 70** 30 55** 45
B-112 35 65 25 75
B-113 40 60 30 70
B-114 62* 38 50* 50
B-306 85** 15 60** 40
B-360 26 74 30 70
B-361 62* 38 40 60
Control 25 25 74
(without treatment) HCP 0,95 12.2 75 22.0
HCP 0,99 22.4 40.4
Note. Hereinafter: * — P < 0.05 relative to control; ** — P <0.01 relative to control.
Table 3. Entomocidal activity (CK50) of Pseudomonas chlororaphis subsp. aureofaciens strains — gaupsin components [7]
Insect species Development stage Strains number
B-306 B-111
Lead-stripped leaf rollers (Ptycholoma lecheana) L 2 1.2 8.7
Meshleaf roller (Adoxophyes orana) L 2 0.6 11.7
Currant curled torturer (Pandemis cerasana) L 3 1.1 16.7
Bunch leaf roller (Lobesia botrana) L 2 0.9 2.0
Plum moth (Grapholita funebrana) L 5 0.4 1.4
Apple moth (Hyponomeuta malinella) L 3 0.4 1.3
Gooseberry fruit moth (Zophodia convolutella) L 4 0.6 10.7
Pudenitsa plum (Angerona prunaria) L 4 0.3 38.3
Colorado beetle (Leptinotarsa decemlineata) L 4 4.2 14.7
Note. CK50 is the number of cells (millions in ml) which causes the 50 % insects mortality
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 non-dialyzed 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 [18-23]. 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 0.001-10.0 mg/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 strains-gaupsin 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
Abacus 1.5 1/ ha Gaupsin 5.0 1/ha Control preparations (without fungicidal treatment) Preparation
H o xs Bottom H o Bottom H o Bottom Tier
10.0±0.4 3.0±0.08* 11.5±0.5 CO ¡S3 If © ¡S3 * 12.5±0.5 7.5±0.4 growth, % Cercosporosis
to 91 If h-1 05 * 25.0±1.6 32.1±1.7 to to o If h-1 05 45.0±2.3 32.0±1.5 spreading, %
20.0±0.8 60.0±3.7 00 o If o 57.3±3.4 efficiency, %
13.3+1.4* CO o If © CO * 13.8±1.2 CO b* If © CO * 15.0±1.2 8.0±0.6 growth, % Ascochitosis
29.8+1.9 CO © o If to ¡S3 35.5±2.3 30.0±1.9 00 ¡S3 If to 25.0±1.5 spreading, %
11.3±1.2 62.5±3.6 00 o If o ^ 56.3±3.3 efficiency, %
00 o If o * * CO w If © CO * * 00 b* If © * ¡S3 If © ¡S3 * * 16.0±1.3 12.5±1.1 growth, % Peronosporosis
h-1 O o h-1 o o h-1 o o h-1 O o h-1 O o h-1 O o spreading, %
50±4.2 72.0+6.8 46.9±3.7 66.4±5.9 efficiency, %
3.0±0.3 to ¡S3 If © CO 3.2±0.3 2.0±0.1 5.2±0.4 3.5±0.4 growth, % Septoriosis
If © 05 * 10.5±1.0* 8.0±0.6 10.0±0.9* 10.1±0.9 16.2±1.3 spreading, %
42.3±3.1 62.9±4.8 38.5±3.3 to b If CO efficiency, %
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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.
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АНТИМ1КРОБНА, ЕНТОМОПАТОГЕННА ТА ПРОТИВ1РУСНА АКТИВН1СТЬ Б1ОПРЕПАРАТУ ГАУПСИН НА ОСНОВ1 ШТАМ1В
Pseudomonas chlororaphis
О. А. КЬпрЬанова1 Л. А. Сафронова1 А .О. Просянов2
Институт мшробмлогй i вiрусологп iM. Д. К. Заболотного НАН Укра!ни, Ки!в 2Товариство з обмеженою вiдповiдальнiстю Компанiя «БмШк», Микола!в
E-mail: safronova_larisa@ukr.net
Метою огляду було подати результати бiльш нiж десятирiчного дослiдження бш-препарату гаупсин, створеного на основ1 двох штамiв Pseudomonas chlororaphis subsp. aureofaciens УКМ В-111 i УКМ В-306, що мае антифунгальну, ентомопатогенну та проти-вiрусну активнiсть. Наведено данi щодо синтезу цими штамами антибiотичних ре-човин — похщних феназину i фешлшролу. У штамiв-компонентiв гаупсину виявлено ен-томоциднi властивостi щодо широкого спектра комах-шкщнишв. На моделi вiрусу тютюново! моза!ки встановлено антивiрусну активнiсть гаупсину, що пов'язана з утворенням термоста-бшьних екзополiмерiв, якi мiстять нейтральш моносахариди. Високоактивними противiрус-ними агентами виявились також лшопол^аха-риди штамiв В-111 i В-306. Встановлено будову 1хшх О-специфiчних полiсахаридiв. Останш структурно гетерогенш, представленi лiнiйни-ми повторюваними три- та тетрасахаридними ланками i мають ранiше не описану специфiч-ну будову.
Ключовi слова: бiопрепарат гаупсин, Pseudo-mo nas chlororaphis subsp. aureofaciens, антибмтики-феназини, пiролнiтрин, ентомо-патогеннi властивоси, противiрусна актив-нiсть.
АНТИМИКРОБНАЯ, ЭНТОМОПАТОГЕННАЯ И ПРОТИВОВИРУСНАЯ АКТИВНОСТЬ БИОПРЕПАРАТА ГАУПСИН НА ОСНОВЕ ШТАММОВ
Pseudomonas chlororaphis
Е. А. Киприанова1 Л. А. Сафронова1 А. О. Просянов2
1Институт микробиологии и вирусологии им. Д. К. Заболотного НАН Украины, Киев 2Общество с ограниченной ответственностью Компания «БиоНик», Николаев
E-mail: safronova_larisa@ukr.net
Целью обзора было представить результаты более чем десятилетнего исследования биопрепарата гаупсин, созданного на основе двух штаммов Pseudomonas chlororaphis subsp. aureofaciens УКМ В-111 и УКМ В-306 и обладающего антифунгальной, энтомопа-тогенной и противовирусной активностью. Представлены данные о синтезируемых этими штаммами антибиотических веществах — производных феназина и фенилпиррола. У штаммов-продуцентов гаупсина обнаружены эн-томоцидные свойства в отношении широкого спектра насекомых-вредителей. На модели вируса табачной мозаики установлена антивирусная активность гаупсина, связанная с образованием термостабильных экзополиме-ров, содержащих нейтральные моносахариды. Высокоактивными противовирусными агентами оказались также липополисахариды штаммов В-111 и В-306. Установлено строение их О-специфических полисахаридов. Последние структурно гетерогенны, представлены линейными повторяющимися три- и тетрасахарид-ными звеньями и имеют ранее не описанное специфическое строение.
Ключевые слова: биопрепарат гаупсин, Pseudomonas chlororaphis subsp. aureofaciens, антибиотики-феназины, пирролнитрин, энто-мопатогенные свойства, противовирусная активность.
