Эффективность применения бактерий Pseudomonas Sp. b-6798 для защиты сельскохозяйственных культур от фитопатогенов в условиях Западной Сибири | Вестн. Том. гос. ун-та. Биология. 2013. № 3 (23).

Эффективность применения бактерий Pseudomonas Sp. b-6798 для защиты сельскохозяйственных культур от фитопатогенов в условиях Западной Сибири

Биологический метод защиты растений от фитопатогенов основан на применении бактерий - антагонистов фитопатогенов. На современных отечественном и зарубежном рынках существует ряд биопрепаратов на их основе, но при этом продолжается поиск более эффективных агентов защиты растений. В работе исследована возможность применения формальдегидрезистент-ных бактерий Pseudomonas sp. В-6798 в качестве потенциальной основы биофунгицида. Для учета антифунгальной активности штамма использовались различные методики, в том числе разработанный авторами метод определения кинетики ингибирования роста фитопатогенных грибов бактериями-антагонистами. Оценка ростостимулирующей активности бактерий проводилась как в лабораторных, так и в полевых экспериментах. В качестве тест-объектов использованы следующие сельскохозяйственные культуры: пшеница, овес, кукуруза, картофель и лен-долгунец. Установлено, что обработка семян бактериями Pseudomonas sp. B-6798 снижает общую пораженность зерновых возбудителями семенных инфекций на 12-36% за счет уменьшения процента семян, пораженных гельминтоспориозно-фузариозными возбудителями корневых гнилей. Бактеризация клубней и растений положительно сказывается на снижении развития заболеваний картофеля: фитофтороза в вегетационный период - на 4550%, ризоктониоза и парши обыкновенной в период хранения - на 40-70%. Оптимальные для развития растений концентрации бактерий находятся в пределах 10-10 КОЕ/мл. Показано, что под действием бактерий в лабораторных тестах происходит увеличение длины проростка в 1,5-3,5 раза, сухой биомассы -1,5-5 раз, суммарной длины корневой системы -1,7-3,0 раза. Согласно результатам полевых испытаний, бактеризация способствует увеличению урожайности зерновых культур и картофеля на 10-40% в зависимости от вида и сорта.

Effectiveness of applying bacteria Pseudomonas sp., strain B-6798, for anti-phytopathogenic protection of crops i.pdf Introduction At the present time, most part of microbial formulations is based on rhizo-sphere bacteria related to genus Pseudomonas. The domestic microbial industry produces numerous biological fungicides using these bacteria, including the following trademarks: such Pseudomonas-based formulations as Pseudobacterin 2™, Agat-25K, Planriz and others [1, 3]. Many formulations based on different species of Pseudomonas genus are known worldwide: Dagger G, Conquer, Victus, BioCoat, Blue Circle, Intercept [4-5]. Wide utilization of Pseudomonas spp. bacteria in biotechnology is linked with multifunctional properties of these bacteria, together with direct inhibition of phytopathogenic microorganisms and as a result of the synthesis of siderophores, cyanide, antibiotics, plant growth stimulation, vitamins, phosphate mobilization and nitrogen fixation [1-5]. Despite a wide range of already developed and applied microbial fungicides, there is a constant search for new strains capable of providing more stable positive effects on plant growth and development and inhibiting phytopathogens [2]. The first experiments linked with estimation of antifungal properties of bacteria Pseudomonas sp., strain В-6798, demonstrated high antagonistic activity of this strain to the most economically important pathogenic fungi. The antagonist is capable of active growth and multiplication in the rhizosphere of agricultural plants. The first data served as a reason for more detailed investigation of useful properties of the new bacterial strain [6-7]. It is known that a stimulating effect on the growth and development of plant vegetative mass is based on the fact that Pseudomonas are capable of synthesizing various growth regulators (indole-3-acetic acid - IAA, gibberellins) and vitamins [1-8]. According to Ju.A. Gushchina and I.F. Glovatskaya [9], bacteria of the strain В-6798 can secrete auxin substances to the environment, particularly, IAA in the quantity of 0.8 mg/ml. The purpose of the present work was connected with further study and summary of the results on the properties of formaldehyde-utilizing Pseudomonas sp., strain В-6798, as a plant growth stimulator and a potential inhibitor of phytopathogenic fungi affecting various crops. Material and methods Research objects The strain of Pseudomonas sp., strain B-6798, was isolated from active silt in purifying construction of Tomsk petrochemical industrial complex. These are facultative methylotrophic bacteria capable of utilizing formaldehyde as a part of poor mineral media as the only source of carbon and energy in the concentration of 250 mmol. Fusarium oxysporum, F. oxysporum f.sp. lini, F. oxysporum f.sp. gladioli, Bi-polaris sorokiniana and Rhizoctonia solani were examined as test objects for assessing antagonistic bacterial activity. Evaluation of Pseudomonas sp., strain B-6798, growth-stimulating activity, was realized basing on the seeds of the following crops: Moldavskaya-215 maize variety (the average seed weight - 0.5 g, total germination - 75%), Tulun-skaya-12 wheat variety (0.03 g, 97%), Narymsky-943 oats variety (0.05g, 93%), Tomsky-10 fiber flax variety, as well as potato tubers of Fresco, Zhukovsky early ripening, Nevsky and Lugovskoy varieties. Media and cultivation. Bacteria Pseudomonas sp., strain B-6798, were cultivated in 50ml of the minimal М9 medium [10] with formaldehyde (2-4 g/l) in a 250-ml retort. Microorganisms were incubated at 30±0.5°С in thermostat until the quantity was established in the limit from 1 to 9*109 CFU/ml. The quantity control was performed by direct counting of cells in a Goryaev's chamber and by inoculation in accordance with the limiting-dilution method on the solid medium with a further count of CFU. 20-% wort agar (4-6°B) was used for phytopathogenic fungi incubation. Investigation of Fungistatic Properties Investigation of Kinetics of Phytopathogenic Fungi Growth Inhibition by Bacteria Analyses of antifungal activity of the strain in laboratory experiments were conducted using both standard method of co-seeding of bacteria-antagonists together with phytopathogenic fungi located on the surface of the medium with a further evaluation of mycelium development inhibition and the method of determining kinetics of phytopathogenic fungi growth inhibition by bacteria-antagonists [6]. The second method elaborated in our laboratory assessed the fungistatic effect according to reduction in the growth rate of fungal colonies on solid medium with different bacteria concentrations, using standardized inoculation procedure. Investigation of the influence of bacterization on seeds contaminated by phy-topathogens. During the experiment, the seeds, previously disinfected with 70% ethanol solution, were soaked in bacterial suspension with concentration 1-6 x 106 CFU/ml for 30 minutes. Control seeds were soaked in sterilized water. The presence of fungistatic effect with experimental strain bacteria was estimated according to reduction in seed infestation by seed infection agents when conducting a phytopathological analysis in sterile absorbent paper rolls 7 days after the setting up of the experiment [11]. Fungistatic effect assessment in field experiments was carried out according to disease incidence and index with plants treated with bacteria in comparison with plants without bacterization [11-12]. The severity of cereal crops infection with root and foot rots was estimated according to the degree of browning of the stem base and plant root system, selected among the control and experimental variants. A rating scale was used to determine plant disease index [11-12]. Root rots were examined three times during the growing season: I - the phase of germination, II - tillering and III - flowering. To evaluate the effect of the bacterial preparation on potato blight development during the growing season, inspections of bacte-rized and control plants were organised. The development of blight affection of plants was analyzed four times: I - during series closing, II - during budding, III - during full blossoming and IV - before top-killing. Potato late blight was estimated in percentage terms on a generally accepted scale [12]. Disease incidence and index were determined by standard methods [11-12]. Disease incidence is a number of diseased plants in percent to total number of plants observed. Disease incidence was calculated as follows: Total number of diseased plants Incidence = -x100. Total number of plants observed Disease index is a measure of disease damage assessment which was calculated in percent as follows: r , (0 x a) + (1 x b) + (2 x c) + (3 x d) + (4 x e) 100 Index =-x-, (a + b + c + d + e) 4 a, b, c, d, e - the number of tillers examined which fall into the categories 0, 1, 2, 3 and 4, respectively To assess bacterial influence on a phytosanitary condition of the new crop during the storage period, a number of phytopathological analyses of potato tubers treated with bacteria and control plants were conducted. At least 100 tubers from each variant were selected three times during the whole period of storage. The tubers were washed, examined and cut in order to identify external and internal symptoms of diseases. Investigation of stimulating activity Investigation of the influence of concentration ofbacterial suspension on plant growth and development in vegetation experiments In the experiments, a model of small terrestrial artificial systems was created, consisting of three parts: a sterile substrate, used to fill vegetation vessels, host-plant and bacterial strains at concentrations of 101-109 CFU/ml. Pre-germinated and disinfected seeds of cereal crops and fiber flax were placed in test tubes with sand (12.5 cm3); one-centimeter long disinfected potato shoots were put in a container with sterile soil and evenly moistened with sterile Knop's solution. Inoculation was carried out by 0.1 ml of bacterial suspension at different concentrations. Plants were grown in a climate camera: 12-hour light period, illumination intensity - 3 000 lux and temperature ±22±1°С. The experiment was carried out in three replications with 35-40 plants per each variant. The experiments being terminated, the following parameters were measured: the main root length (for maize), total root system length (for oats and wheat), the number of second-order roots and plant dry biomass. For each potato plant was measured the wet vegetation mass, the number of leaves and the number of tubers together with their weight. Investigation of the influence ofbacterial culture on plant growth and development in field experiments Field experiments lasted for 6 growing seasons. The soil in the experiments was gray, forest, podzolized and medium-loam with low humus content (4-5%) as well as a thin humus accumulation horizon (20-25 cm). Maize seeds were planted at a distance of 10-15 cm from each other, wheat and oats were sown at 600 seeds/m2, and potatoes were planted at 42000 tubers/ ha. Bacterisation of seeds was conducted with the help of pre-planting humidifica-tion treatment by bacterial culture with a titre of no less than 106 CFU/ml. Potato tubers were soaked in bacterial suspension of the same concentration for 30 minutes. Seeds and tubers treated with water in the same manner as seeds in the test variants were used as control. In the period of the experiment, the length of the maximum potato shoot was measured in each plant in all variants during a month since sprouting. The number of stems and the intensity of plant flowering were also analyzed during the experiments. Small sheaves of other plants were selected uniformly over the whole area of the test variant. Then, the length and dry biomass of the selected plants were measured; the quantity of grains per filled ear, the number of reproductive stems and the weight of 1 000 grains were examined in cereal plants during the phase of full ripeness. Processing experimental results The data obtained during the experiments were processed by means of StatSoft STATISTICA 6.0. The data are presented as a mean with confidence interval, using Student's t-test for 95% significance level. Statistical significance of the obtained results of seed phytopathological expertise, the incidence of root rots and foliar infections in field experiments were assessed by comparing sampling fractions, using Student's t-test for the probabilities 25-75% inclusive and using F-test for other probability values. Growth rates of fungal colonies on solid medium were determined by linear regression analysis. The data on parameters of plant growth and development in field experiments were compared by non-parametric Mann-Whitney test (р < 0.05) [13]. Results and discussion Antifungal activity of Pseudomonas sp., strain В-6798, in model experiments In vitro experiments, the strain showed a high antagonistic activity against a number of fungal pathogens. The bacteria facilitated reduction in the growth rate of fungal colonies on solid media in Petri dishes within the limits of 5-8 times in comparison with control (fig. 1). In control, fungal colonies grew on a bacteria-free medium. It was experimentally established that the magnitude of fungistatic effect depended on the type of phytopathogens and the concentration of bacteria in dishes. Fungus Bipolaris sorokiniana was the most sensitive to antagonistic bacteria and F. oxysporum f.sp. lini - the least. The ratio of antagonist concentration to the level of disease development inhibition is very important for development and application of microbial fungicides. G.M. Raaijmakers et al [14] demonstrated that there was a ''threshold" level of antagonist concentration, below which a slight decrease in bacteria concentration, expressed as a logarithm (log10-dose), results in a significant modification of inhibition of the development of fungi pathogens. Our experiments detected no threshold effects in relation to the growth rate of phytopathogenic fungal colonies. In order to describe kinetics of fungal colonies growth inhibition by the studied bacteria, we used N.D. Ierusalimsky modified equation [6]. Fungal colonies growth inhibition was already fixed at bacteria concentration 101 cells/ml in the inoculum. As bacteria concentrations in Petri dishes increased, the growth rate of fungal colonies tended to reach the limit value, not equal to zero, i.e. no total absolute fungal growth inhibition by bacteria was observed. In all the studied relationships between fungi and bacteria, concentration of 102-103 cells/ml is enough to reach the smallest limit value of fungal growth rate. 12 3 The influence of bacterization on seeds to infection agents Phytopathological examination of bacterized seeds of wheat and oats revealed an antagonistic effectofbacteria onseed infectionagents in plantamodQl conditions. 60 55 50 • 45 • 40 • cs % 35 30 • 25 • Control Bnatctizntidg Bnatctizntidg Fig.2. Totalinfestation ofwheatseedsbyseed infectionagentsduringseedbacterization Note. * - statistically significantdifferencefromthecontrolvariant(p 0.05). This is due to the fact that this variety is late blight resistant and late blight is not widely dispersed on this variety, even in control. Plant systemic resistance formation during bacterization can be also demonstrated by the results of a phytopathological analysis of wheat seeds (fig. 5). —I-1-1-1— 45 40 35 30 Control Bacterization o4 £ 25

Ключевые слова

фитопатоген, ростостимулирующая активность, биофунгицид, бактерии-антагонисты, антифунгальная активность, phytopathogen, growth-promoting activity, biofungicide, bacteria-antagonists, antifungal activity

Авторы

ФИООрганизацияДополнительноE-mail
Акимова Елена ЕвгеньевнаТомский государственный университет; Сибирский научно-исследовательский институт сельского хозяйства и торфа Российской академии сельского хозяйства (г. Томск)кандидат биологических наук, старший преподаватель кафедры экологической и сельскохозяйственной биотехнологии Биологического института; старший научный сотрудник лаборатории биотехнологииbiotech@sibmail.com
Минаева Оксана МодестовнаТомский государственный университеткандидат биологических наук, доцент кафедр экологической и сельскохозяйственной биотехнологии и цитологии и генетики Биологического институтаmom05@mail.ru
Всего: 2

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 Эффективность применения бактерий <i>Pseudomonas</i> Sp. b-6798 для защиты сельскохозяйственных культур от фитопатогенов в условиях Западной Сибири | Вестн. Том. гос. ун-та. Биология. 2013. № 3 (23).

Эффективность применения бактерий Pseudomonas Sp. b-6798 для защиты сельскохозяйственных культур от фитопатогенов в условиях Западной Сибири | Вестн. Том. гос. ун-та. Биология. 2013. № 3 (23).