Phytopathogenic bacteria and phytoplasmas, in particular Pseudomonas syringae pv. atrofaciens and Acholeplasma laidlawii var. granulum are causative agents of cereal crops diseases. Nanoparticles and biologically active compounds created with the use of nanotechnologies are considered as alternative modern measures to control phytopathogenic microorganisms. Antimicrobial activity is already established for gold, silver, copper, magnesium, iron, zinc, aluminum, cerium, titanium, cadmium, nickel and vanadium. At the same time, antimicrobial activity of germanium and its citrates as well as citrates of others nanoparticles, including vanadium, are studied insufficiently. The physiological effect of vanadium and germanium nanoaquacitrates on wheat plants in vivo, both on state and photochemical activity of the photosynthetic apparatus and activity of superoxide dismutase of plant tissue are poorly known too. Aim. The aim of our work was to study the antimicrobial activity of vanadium and germanium nanoaquacitrates in vitro and their physiological influence on wheat plants in vivo. Methods. A. laidlawii var. granulum 118 (UCM BM-34) was cultured in the liquid nutrient medium CM IMV-72 (pH=7.8) in thermostat at 320 C for 72 hours. P. syringae pv. atrofaciens D13 was cultured on potato agar in thermostat at 26–280°C. For artificial infection a bacterial suspension with a density of 1×109 CFU/ml according to the turbidity standard was prepared. Nanoaquacitrates of V (30 mg/dm3) and Ge (5 mg/dm3) were used in our study. After a short-term (15 minutes) action of the test compounds, morphological features of A. laidlawii var. granulum and P. syringae pv. atrofaciens were studied using a transmission electron microscope (TEM) JEOL JSM 1400 (Zabolotny IMV NASU). The plants of wheat of Pecheryanka variety were grown in field condition on 50 m2 area plot. Pre-sowing treatment of spring wheat seeds before sowing was carried out with solutions of V (30 mg/dm3) and Ge (5 mg/dm3) nanoaquacitrates according to the experimental scheme. The experiment was carried out in three repetitions. In addition, biological preparation (B.P.) Extrakon, which consists from natural consortium of beneficial microorganisms inoculated into a peat-like substrate, was applied into the soil before sowing. Artificial inoculation by phytoplasma A. laidlawii var. granulum 118 was performed using Clement`s method (subepidermal injection into the stem) in the tillering phase of wheat plants growth. Artificial infection of wheat plants with P. syringae pv. atrofaciens D13 was performed by injecting a bacterial suspension into the stem in the booting phase of plants growth. The activity of superoxide dismutase (SOD) was determined by the ability of the enzyme to inhibit the photochemical reduction of nitro blue tetrazolium. Changes in the following fluorescent parameters were analyzed: Fv/Fp and Rfd. For statistical processing of data, calculations of the arithmetic mean and its standard error were performed. Results. It was shown using electron microscopy that contact of Ge and V nanoaquacitrates with P. syringae pv. atrofaciens D13 and A. laidlawii var. granulum 118 resulted in cytomorphological changes of cells, homeostatic disruption and death. It was found in the field studies that SOD activity level increased in the tissues of wheat leaves to different values in plants infected with phytopathogenic microorganisms without any treatment and in cases of pre-sowing treatment with Ge and V nanoaquacitrates and B.P. Extrakon. Field investigations showed that combined use of consortium of soil microorganisms (B.P. Extrakon) and pre-sowing treatment of wheat seeds with V and Ge nanoaquacitrates decreased negative impact of infection caused by A. laidlawii var. granulum 118 and P. syringae pv. atrofaciens D13 on the state and photochemical activity of the photosynthetic apparatus of Triticum aestivum: the level of quantum efficiency of photosystem II (PSII) and the assimilation activity coefficient Rfd increased. This effect of studied biologically active substances is due to both antimicrobial action and activation of the antioxidant defense system of plant cells. Conclusions. It was shown in our investigations that SOD activity increased in leaf tissues after application of biologically active agents in the next order: pre-sowing treatment with Ge nanoaquacitrates < pre-sowing treatment with V nanoaquacitrates < biological product (B.P.) Extrakon + pre-treatment with Ge nanoaquacitrates < B.P. Extrakon + pre-sowing treatment with V nanoaquacitrates. A significant increase of SOD activity of wheat leaves (in the tubing phase) was noticed at both types of infection – caused by phytopathogenic bacteria and phytoplasma, especially for the last one. A significant increase in the surface area of assimilation apparatus of wheat plants (in the tillering phase) in cases of wheat seeds pre-sowing treatment with Ge and V nanoaquacitrates before planting was observed. Pre-sowing treatment of wheat seeds with Ge and V nanoaquacitrates in combination with B.P. Extrakon use on wheat plants infected with A. laidlawii var. granulum and P. syringae pv. atrofaciens improved photosynthesis and status of photosynthetic apparatus of plants.
Electron microscope investigations have revealed disintegration of the cell wall of Pseudomonas syringae pv. atrofaciens and subsequent killing of bacterial cells after application of 1% solutions of the citrates of Ag-Cu nanoparticles, the complex Co-Cu-Zn-Fe-Mn-Mo-Mg (Avatar-1) and iodine-selenium. In field conditions, it was revealed that pre-sowing treatment of wheat seeds with the aforementioned 1% solutions significantly inhibited the development of basal glume rot in cereals with artificial infection of Pseudomonas syringae рv. аtrofaciens. Consequently, reduction of the typical symptoms of basal glume rot of cereals and the percentage of isolated cells of Pseudomonas syringae рv. аtrofaciens D13 were detected in wheat plants upon pre-sowing treatment of wheat seeds by experimental biocides. These results can be explained by structural changes in the bacterial cells of the pathogen that lead to reduction of viable forms of bacteria due to damage of both the cell wall and internal contents.
In field experiments, increase the quantum efficiency of PSII (Fv/Fp), the Rfd-values (the chlorophyll fluorescence vitality indices) and the content of photosynthetic pigments in wheat leaves at pretreatment of wheat seeds with solutions of citrate nanoparticles has been shown. The green weight of plants at bacterial and phytoplasmas infecting at pretreatment with solutions of citrate nanoparticles, was increase, than without pre-treatment, in order: GeNPs>I-Se>SeNPs>VNPs as it has been shown. The Fv/Fp-values in leaves infected with phytoplasmas of plants at pretreatment of VNPs or I-Se solutions were more, than without pretreatment. The Fv/Fp-value in leafs with bacterial infecting at pretreatment of GeNPs with compared to infected plants without pretreatment was increased has been shown. The Rfd-value in leaves at pre-sowing treatment of GeNPs or I-Se and bacterial infecting compared to infected plants without the pr-treatment was more has been shown. This value at infected plants with phytoplasmas at pretreatment of GeNPs or VNPs, compared to infected plants without pretreatment, was increased. In greenhouse experiments, Fv/Fp-values of plants leaves infected of phytoplasmas, bacteria and wheat streak mosaic virus (WSMV) at pre-treatment of solutions of citrate nanoparticles compared to infected plants without pretreatment has been increased. The pretreatment with nanoparticles GeNPs caused grain production by 15.6% increased has been shown. The weight grains/10 in plants on variant with pretreatment with Ge
The influence of pre-sowing seed treatments of selenium nanocitrates (SeNPs) and application of soil formed microorganism consortium (biological preparation (BP) Extrakon) on wheat plants infected with wheat streak mosaic virus (WSMV) were investigated in greenhouse and fields conditions in 2018-2019 (on territories of Zabolotny Institute of Microbiology and Virology of NASU).The pre-sowing seed treatments of 1% selenium nanocitrates (SeNPs) with application of BP Extrakon initiating the growth of juvenile wheat in laboratory experiments were found.In the field experiment on variants of WSMV-infected plants, two weeks after inoculation (tillering stage), we observed the appearance of characteristic symptoms of yellow mosaic with intermittent strokes on the leaves. In the boot phase, a lag in the growth of WSMV-infected plants compared with intact plants was already clearly visible, which was confirmed by the ELISA test.It was shown, increase of the quantum efficiency of PS II (F v /F p ) of WSMV-infected plants at pre-sowing treatment of 1% SeNPs and application of microbial consortium (BP Extrakon) compared with virus-infected plants without treatment.In plant tissue of all experimental variants increase activity of superoxide dismutase was shown. It was shown more significant increase of the SODactivity on the variant of WSMV-infected plants with application of BP Extrakon
In greenhouse experiments changes in several metabolic parameters soybeans plants after inoculation by P. savastanoi pv. glycinea 9190, X. axonopodis pv. glycines 9192, BCMV, A. laidlawii on background pre-treatment by nanochelates (NHs) of V, Ge, Cu, Mo have been shown. The phenolic compounds content increased in leaves at inoculation by phytopathogens on background pre-treatment by NHs and without it. The content of H 2 O 2 in leaves increased in variants: BCMV (+VNHs, GeNHs, CuNHs), 9192 (+VNHs, CuNHs), 9190 (+GeNHs, CuNHs), A. laidlawii+MoNHs. But it decreased in the variants: BCMV + MoNHs, A. laidlawii + VNHs, 9190 + VNHs, 9192 (+ GeNHs, CuNHs) compared to controls. The peroxidase activity increased in variants: A. laidlawii, 9190, GeNHs + 9190, GeNHs + 9192, CuNHs, CuNHs (+ phytoplasmas, 9192), MoNHs (+ BCMV, phytoplasmas, 9190, 9192) with decreased catalase activity. The content of ABA in the leaves increased in variants: 9190, 9192 (+ VNHs, CuNHs), GeNHs + 9190, but the IAA content reduced. However, on variants MoNHs (+9190, 9192), GeNHs + 9192, A. laidlawii, A. laidlawii (+ GeNHs, CuNHs, MoNHs) the content of both phytohormones was decreased. The content of IAA and ABA in BCMV-infected leaves without treatment was increased, but on background of NHsit decreased
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