マルチウェルプレートを用いたムギ種子の簡便な黒節病菌保菌粒率調査法

Diatomaceous earth low-lethal dose effects on the fitness of entomopathogenic fungus, Beauveria bassiana, against two coleopteran stored product pests

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.

Globally, basal kernel blight caused by Pseudomonas syringae pv. syringae (PSS) has been recognized as the most serious diseaseo of barley. Recently, polymerase chain reaction (PCR) based gene sequencing is a more powerful tool to identify bacterial pathogens from seeds as well as other plant materials. The disease can be controlled by chemical treatment in the field which has harmful consequences on the environment and biodiversity. The application of plant extracts for the eco-friendly management of seed-borne pathogens is a popular new biocontrol method. Therefore, this study aimed at isolating and identifying PSS from seeds of barley and assessing the antimicrobial potential of the extracts of some plants viz., Citrus limon, Hibiscus rosa-sinensis, Ricinus communis, Psidium guajava, Murraya koenigii, Withania somnifera, Eucalyptus globulus and Tecomella undulata to promote effective biocontrol strategy. A total of 28 bacterial pathogens were isolated and tested for various biochemical tests. Results of the LOPAT test (+---+) and GATTa test (++--) primarily indicated the presence of PSS. The 16S rRNA gene sequencing was performed on PCR-amplified products with 8F and 1492R primers. The two isolates HV 03 and KASS showed 99.8 and 99.6% homology with strain PSS strain KFB 86 which was isolated from Ficus carica in Serbia. The highest antibacterial activity in vitro was obtained with methanol extract of W. somnifera, P. guajava, E. globulus and M. koenigii. The results specified that the plant extracts which showed promising antibacterial activity may be considered an effective tool for the management of disease caused by PSS in barley.

In this study, radio-frequency (RF) plasma was used to treat the surface seeds of wheat and barley. Plasma treatment was conducted for different exposure times (1, 5, and 10 min) and at different powers (20, 50, 80 and 100 W), keeping the air gas pressure fixed at 1.0 torr. It was observed that the seeds that were exposed to 80 W of plasma power for 10 min. exhibited a higher water absorption rate. The weight gain of the treated wheat and barley seeds was increased by 62% and 12%, respectively, compared to the control sample (untreated seeds). Spectroscopic and microstructural analyses of the seed surface indicated that the plasma treatment caused chemical etching, which improved the water uptake of the treated seeds.

Vibrating separation on sieves is one of the most common ways of loose materialsseparating, and seeds of agricultural crops in particular. Vibrating separators have a number of advantages, as they allow combining the processes of transportation and material separation. In the designs of the existing seed cleaning machines of this type, from the point of view of the design parameters of the working body, setting and mode of operation, it is difficult to improve the indicator of the completeness of the separation of grain mixtures into fractions. Therefore, there is a need to find ways to intensify this process, to develop and study the operation of technical means for their implementation. One of the ways to improve the conditions for the movement of components of grain mixtures through the holes of the sieves is to create an electric field above them. Theoretically, there is a certain initial value of the intensity of the electric field, at which the seed will start to orient itself in it. The optimal tension value will be the one at which the degree of orientation of the seeds on the oscillating sieve in the longitudinal direction will be maximum (when their longitudinal axis will be perpendicular to the sieve). The paper describes the results of the process study of separating seeds of cereal crops on a vibrating electric separator and substantiates the optimal values of the electric field intensity E, which can achieve the maximum effect of seed orientation on the sieve. The results of experimental studies showed that the effect of the electric field on the seeds of different crops is not the same. It reveals itself the least on wheat seeds, since the quantity of seeds oriented in the electric field was about 35%. For barley seeds, the value of the studied indicator was 48%. The maximum percentage of oriented oat seeds was about 53% at the maximum intensity of the electric field of 6.67 kV/cmover the sieve. It was revealed that the orientation of the seeds on the sieve affects the intensity of its sieving through the holes. Under conditions of maximum orientation, the index of sifting of wheat seed increased by 20...23%, barley seed - by 26...30%, oats seed - by 47%.

Achieving high crop yields in agriculture depends largely on the quality of seeds and planting materials. Seed-borne infections reduce germination capacity and overall plant productivity, highlighting the need for treatments that combine growth-stimulating and fungicidal properties. This study aimed to evaluate the stimulant and antifungal effects of aqueous solutions of dithiocarbamates on wheat and barley seeds, identify optimal concentrations, and assess their impact on seed quality and pathogenic microflora. Results and Discussion. Seeds of wheat and barley were treated with five dithiocarbamate-containing preparations at concentrations of 0.001%, 0.01%, and 0.1%. Germination energy, laboratory germination, seedling growth intensity, microbial activity, and percentage of infected seeds were assessed. At a concentration of 0.001%, wheat seeds treated with Preparations 1 and 3-5 showed high germination rates (94.0-96.6%), although infection levels varied (6.0-27.3%). In barley, Preparations 2-5 demonstrated significant stimulation of germination (93.0-98.0%) and reduced seed infection to 8.0-22.0%. The most pronounced antifungal activity in barley was observed for Preparations 1 and 3, reducing infection to 10.0-12.0% and 9.0-11.3%, respectively, compared to 77.3% in the control. In wheat, Preparations 3 and 5 effectively reduced infection rates to 10.6-19.0% and 6.0- 16.0%, respectively. Conclusion. The results suggest that dithiocarbamate-containing preparations can enhance seed viability while limiting pathogen development. Two compounds were most effective: Preparation 5 (sodium benzylmethylcarbamodithioate) for wheat and Preparation 3 (sodium dibenzylcarbamodithioate) for barley. These findings support their potential use as dual-action seed treatments in cereal crop production.

The closeness of wells in multiwell tissue culture plates makes it difficult to remove individual ones without distributing the adjacent wells. Moreover, critical point drying frequently introduces artifact into the culture monolayer grown on plastic substrate. These problems make it difficult to process such cultures for scanning electron microscopy. However, for cell kinetic and correlative morphological studies on primary cultures derived from 7,12-dimethylbenz(alpha)anthracene(DMBA)-induced mammary tumors, we have found that Falcon 24-well tissue culture plates are excellent for maintenance of cells and are convenient to use. By plating the cells in alternating, diagonally disposed wells and while the cells are still in the buffer, individual wells can be cut from a multiwell plate without disturbing the cells in adjacent wells. The isolated wells can be used to carry out scanning electron microscope preparation. The height of the well is reduced with a scalpel prior to critical point drying; the remaining well is useful as a handle in mounting the dried sample to stubs for subsequent coating and viewing. Critical point drying and coating of monolayer samples on Falcon plastic are described. The results do not suggest that any artifacts are introduced in our preparations.

Mineral fertilizers which can significantly increase crop productivity have an adverse effect on the soil and the environment as a whole when used for a long time. Microorganisms, as an alternative to mineral fertilizers, stimulate plant growth and development due to their ability to fix nitrogen, produce siderophores, phytohormones and enzymes, dissolve inaccessible elements of mineral nutrition, suppress plant pathogens, and increase consumption of water and nutrients. However, the effectiveness of such preparations highly depends on factors of a new environment. We believe that bacteria from soils that have long been exposed to various agricultural practices may be good plant stimulants. In the presented study, for the first time, we have isolated local active strains of nitrogen-fixing and phosphate-mobilizing bacteria from soils subjected to 74-year stationary intensive farming in the conditions of the Russian Far East and revealed isolates and their combinations which stimulate wheat and barley seed germination and seedling growth. The aim of the work was to study the plant-stimulating properties of nitrogen-fixing and phosphate-mobilizing bacteria from soils that have been actively exposed to mineral fertilizers for a long time. Bacteria were isolated from soils sampled in October 2015 (the experimental field 8, Federal Research Center for Agrobiotechnology FEB RAS, Ussuriysk, Primorsky Territory, Russia). The seeds of wheat Triticum aestivum L. cultivar Primorskaya 50, and barley Hordeum vurlage L. cultivar Tikhookeanskii (collection of the Federal Research Center for Agrobiotechnology FEB RAS) were treated. Of 68 bacterial isolates with different cultural and morphological properties, three isolates, the Acinetobacter spp. N1, Azotobacter spp. N2, and Clostridium spp. N3 were nitrogen fixers, and four isolates, the Serratia spp. P6, Bacillus spp. P7), Arthrobacter spp. P8, and Pantoea spp. P19 were phosphate-mobilizing bacteria. Tests with the monocultures of nitrogen-fixing and phosphate-mobilizing isolates and their different binary compositions showed a 13-51 % increase (p ≤ 0.05) in wheat seed germination energy and 15-54 % increase (p ≤ 0.05) in barley seed germination energy compared to the untreated control. Laboratory germination of wheat seeds increased by 2-32 %, barley seeds by 7-30 % compared to untreated control. The barley seedlings were 1.8 times longer, and the roots were 2.7 times longer. Th binary combination N2P19, P6P19, and P8P19 caused the highest height of seedlings (120-140 mm, p ≤ 0.05), and with P6P7, N2P19, and P6P19 the roots were the longest (120-130 mm, p ≤ 0.05). These results allow us to conclude that short-term soaking seeds in the suspensions of the tested nitrogen-fixing and phosphate-mobilizing isolates improves seed germination energy and laboratory germination, and increases shoot and root length. Binary bacterial compositions have a greater effect on seed germination than monocultures. The strains N1 (Acinetobacter spp.), N2 (Azotobacter spp.), and P19 (Pantoea spp.) are the best stimulants. Species-specific differences in plant response to the treatment is probably due to lack of genetic, biochemical and physiological complementarities between specific plant species and the bacteria.

First experimental results on OCT imaging of internal structure of plant tissues and in situ OCT monitoring of plant tissue regeneration at different water supply are reported. Experiments for evaluating OCT capabilities were performed on Tradescantia. The investigation of seeds swelling was performed on wheat seeds (Triticum L.), barley seeds (Hordeum L.), long-fibred flax seeds (Linum usitatissimum L.) and cucumber seeds (Cucumis sativus L.). These OCT images correlate with standard microscopy data from the same tissue regions. Seeds were exposed to a low-intensity physical factor-the pulsed gradient magnetic field (GMF) with pulse duration 0.1 s and maximum amplitude 5 mT (4 successive pulses during 0.4 s). OCT and OCM enable effective monitoring of fast reactions in plants and seeds at different water supply.

Phytopathogenic bacteria represent a risk to global food production by impacting a variety of crops. The aim of this study was to characterize the contents of bioactive constituents in extracts from Ginkgo biloba L. leaves and fruits and test their activity against six phytopathogenic bacteria that affect horticultural crops. Gas chromatography–mass spectrometry (GC−MS) was used for the chemical profiling of the aqueous methanol extracts, and their bacteriostatic activity against Clavibacter michiganensis subsp. michiganensis, Pseudomonas cichorii, Pseudomonas syringae pv. pisi, Pseudomonas syringae pv. syringae, Pseudomonas syringae pv. tomato, and Xanthomonas vesicatoria (formerly Xanthomonas campestris pv. vesicatoria) was determined in vitro through the agar dilution method. The protective effect of the leaf extract was tested in vivo against the most relevant bacterial pathogens based on their economic/scientific importance, i.e., C. michiganensis subsp. michiganensis and P. syringae pv. pisi, in tomato (Solanum lycopersicum L.) and pea (Pisum sativum L.) plants, respectively, under greenhouse conditions. The GC−MS characterization of G. biloba extracts revealed the presence of dihydro-4-hydroxy-2(3H)-furanone, 2,4-dimethyl-3-hexanol, catechol, 3-O-methyl-D-fructose, 4,6-di-O-methyl-α-D-galactose, methyl 2-O-methyl-α-D-xylofuranoside, and 3-methyl mannoside. In vitro growth inhibition tests showed that, while the fruit extract had no activity, the leaf extract exhibited minimum inhibitory concentrations between 500 and 1000 μg mL−1, which may be attributed to the presence of 2,4-dimethyl-3-hexanol and catechol. In vivo tests of the leaf extract demonstrated full protection in tomato and pea plants at 1000 and 1500 μg∙mL−1, respectively. The results indicate that G. biloba leaves may be employed as a biorational source for integrated pest management in horticulture.

A series of head-to-head bis-benzimidazole and bis-benzoxazole derivatives was synthetized. Their antibacterial activity was examined in planta against Pseudomonas syringae pv. tabaci, the causal agent of wild fire in tobacco and in vitro against Pseudomonas syringae pv. tabaci and Pseudomonas syringae pv. tabaci. All synthetized compounds did not inhibit bacterial growth in vitro. However, they protected Nicotiana benthamiana against P. syringae pv. tabaci in the greenhouse. Only one foliar application at 50 μg ml−1 reduced diameter of leaf lesion by 25 to 52% as well as bacterial population in planta by 0.4 to 0.9 logarithmic units. This protection was associated with the inhibition of the accumulation of H2O2in planta and enhancement of the activity of catalase, ascorbate peroxidase and guaiacol peroxidase. These results suggest that the bis-benzoimidazole and bis-benzoxazole derivatives act as antioxidants and may be used to protect plants against bacterial diseases.

Wildfire leaf spot caused by Psuedomonas syringae pv. tabaci + is a important seedbed diseases of Solanaceous crops in Zimbabwe. It results in loses of economic significance in yield quality every season. The objective of this study was to evaluate the efficacy of Acibenzolar-S-methyl (ABM; Actigard 50 WP, Syngenta Inc.) against Wildfire (Pseudomonas syringae pv. tabaci+) in paprika seedlings. ABM was evaluated for its ability to suppress Pseudomonas syringae pv. tabaci tox + of paprika (Capsicum anuum L.) seedlings in the greenhouse. Four ABM spraying treatments differing in concentration and application intervals were evaluated and compared with the standard practice of spraying copper oxychloride at a rate of 1.56 g ai m-2. Paprika seedlings sprayed with ABM were significantly protected from wildfire (Pseudomonas syringae pv. tabaci tox +). The ABM restricted the incidence and severity of wildfire pathogens from progressing within leaf lamina as shown by a lower Area Under Disease Progress Curve (AUDPC) for disease incidence in seedlings sprayed with ABM when compared to seedlings sprayed with copper oxychloride. Since, both levels of the active ingredient confer resistance exhibiting no significant differences in severity and incidence of wildfire, it is therefore more economic to apply lower rates at lower frequencies in the nursery. The ABM may be a useful tool in IPM programs or replacement of pesticide such as copper oxychloride in the management of Pseudomonas syringae pv. tabaci tox +.

Twenty strains of Pseudomonas syringae pv. tomato were examined for the presence of plasmid DNA. P. syringae pv. tomato plasmids were grouped into five size classes: class A ranged from 95 to 103 kilobases (kb); class B ranged from 71 to 83 kb; class C ranged from 59 to 67 kb; class D ranged from 37 to 39 kb; and class E was 29 kb. All strains contained at least two plasmids in classes A and B. The conjugative ability of P. syringae pv. tomato plasmids in three strains was demonstrated by mobilization of the nonconjugative plasmid RSF1010 into Pseudomonas syringae pv. syringae recipients. Plasmids from the three conjugative strains were labeled with Tn5. Four conjugative plasmids were identified by their repeated transfer to P. syringae pv. syringae recipients. P. syringae pv. tomato strains varied in sensitivity to copper sulfate (CuSO4): MICs were 0.4 to 0.6 mM for sensitive strains, 1.2 mM for moderately resistant strains, and 1.6 to 2.0 mM for very resistant strains. One very resistant strain, PT23, functioned as a donor of copper resistance. Recipient P. syringae pv. syringae strains PS51 and PS61 were inhibited by 0.1 mM CuSO4, whereas the CuSO4 MICs for transconjugant strains PS51(pPT23A) and PS61(pPT23C) were 1.8 and 2.6 mM, respectively. P. syringae pv. tomato strains PT12.2 and PT17.2 were inhibited by 0.6 mM copper sulfate, but their copper sulfate MICs were 2.6 and 1.8 mM, respectively, when they acquired pPT23C. Therefore, copper resistance in PT23 was controlled by two conjugative plasmids, designated pPT23A (101 kb) and pPT23C (67 kb).

Viburnum bacterial blight weakens the growth of Viburnum trees (bushes) and inhibits the physiological processes caused by the Pseudomonas syringae pv. viburni bacterium which survives in the affected stem tissue, plant remains, and soil. The purpose of the study was to examine the bioecological features of the manifestation of Pseudomonas syringae pv. viburni and development of measures to control bacterial leaf spotting in viburnum gardens. During the experiment, diagnostic methods were used to select plant leaves, identify, record, and analyse the affected leaves of viburnum plants by the Pseudomonas syringae pv. viburni bacterium. A method to avoid or reduce the risk of bacterial blights was developed. It was determined that various approaches can be used to prevent bacterial diseases in plants of the Viburnum L. genus, such as selecting more disease-resistant varieties, collecting and destroying fallen leaves and branches after pruning, and following agricultural techniques and gardening practices. For chemical control, copper-based bactericidal preparations, such as copper hydroxide or copper sulfate can be used, which are recommended for use in autumn and spring before budding. Performing these actions will help to prevent the manifestation of bacterial diseases in plants. It is proved that in the conditions of the Northern Forest-Steppe of Ukraine in the system of fruit gardening, it is advisable to grow high- and medium-resistant genotypes of Viburnum vulgaris of the Institute of Horticulture of the National Academy of Agrarian Sciences of Ukraine against bacterial leaf spotting. The practical value of the study lies in the fact that information about bacterial blight or bacterial spotting of viburnum leaves was expanded; it was proved that various species of the Viburnum L. genus differ in their susceptibility to Pseudomonas syringae pv. viburni bacterial damage; it is confirmed that the susceptibility of viburnum plants to this disease can be substantially reduced due to low-susceptible and resistant varieties and species of the Viburnum L. genus and timely technical and chemical measures

Bacterial blight caused by Pseudomonas syringae pv. pisi is an important, but sporadic, disease of field peas (Pisum sativum) in Australia. The presence of P. syringae pv. pisi reduces the profitability of peas due to yield loss and, in some cases, it also limits Australia’s export of peas to some countries. Pseudomonoas syringae pv. pisi is primarily a seed-borne pathogen, but infected pea trash can be an important source of inoculum. Alternative hosts and soil are not regarded as epidemiologically important sources of inoculum. P. syringae pv. pisi survives, multiplies and spreads epiphytically in pea crops. Epiphytic populations of P. syringae pv. pisi only become pathogenic following crop damage caused by frost or severe weather conditions. Frost damage is especially important because the ice nucleating activity of P. syringae pv. pisi initiates frost damage at higher temperatures than occurs in the absence of the bacterium. In addition early-sown crops are more prone to damage from bacterial blight than crops sown later in the season. Pseudomonas syringae pv. pisi consists of seven identified races. One of these (Race 6) lacks all avirulence genes and is common around the world and in Australia. Globally, Race 2 and Race 6 predominate; however, in Australia, Race 3 predominates due to the widespread cultivation of cultivars susceptible to Race 3, but resistant to Race 2. Resistance to Race 6 within P. sativum has not been found but attempts are being made to incorporate a race non-specific resistance identified from P. abyssinicum into field pea. Bacterial blight can be successfully controlled using an integrated disease management strategy incorporating crop rotation, pathogen-free seed, avoidance of planting in areas prone to frequent frosts or extreme wet weather, crop hygiene and avoiding early sowing. Seed treatment and application of foliar bactericides have limited use in control of this disease.