Biosynthesis of Silver Chloride Nanoparticles by Rhizospheric Bacteria and Their Antibacterial Activity against Phytopathogenic Bacterium Ralstonia solanacearum

Ralstonia solanacearum is the most destructive pathogen, causing bacterial wilt disease of eggplant. The present study aimed to develop green synthesis and characterization of silver chloride nanoparticles (AgCl-NPs) by using a native bacterial strain and subsequent evaluation of their antibacterial activity against R. solanacearum. Here, a total of 10 bacterial strains were selected for the biosynthesis of AgCl-NPs. Among them, the highest yield occurred in the synthesis of AgCl-NPs using a cell-free aqueous filtrate of strain IMA13. Ultrastructural observation revealed that the AgCl-NPs were spherical and oval with smooth surfaces and 5–35 nm sizes. XRD analysis studies revealed that these particles contained face-centered cubic crystallites of metallic Ag and AgCl. Moreover, FTIR analysis showed the presence of capping proteins, carbohydrates, lipids, and lipopeptide compounds and crystalline structure of AgCl-NPs. On the basis of phylogenetic analysis using a combination of six gene sequences (16S, gyrA, rpoB, purH, polC, and groEL), we identified strain IMA13 as Bacillus mojavensis. Three kinds of lipopeptide compounds, namely, bacillomycin D, iturin, and fengycin, forming cell-free supernatant produced by strain IAM13, were identified by MALDI-TOF mass spectrometry. Biogenic AgCl-NPs showed substantial antibacterial activity against R. solanacearum at a concentration of 20 µg/mL−1. Motility assays showed that the AgCl-NPs significantly inhibited the swarming and swimming motility (61.4 and 55.8%) against R. solanacearum. Moreover, SEM and TEM analysis showed that direct interaction of AgCl-NPs with bacterial cells caused rupture of cell wall and cytoplasmic membranes, as well as leakage of nucleic acid materials, which ultimately resulted in the death of R. solanacearum. Overall, these findings will help in developing a promising nanopesticide against phytopathogen plant disease management.

Evaluation of Three Antimicrobial Peptides Mixtures to Control the Phytopathogen Responsible for Fire Blight Disease

Fire blight is a severe bacterial plant disease that affects important chain-of-value fruit trees such as pear and apple trees. This disease is caused by Erwinia amylovora, a quarantine phytopathogenic bacterium, which, although highly distributed worldwide, still lacks efficient control measures. The green revolution paradigm demands sustainable agriculture practices, for which antimicrobial peptides (AMPs) have recently caught much attention. The goal of this work was to disclose the bioactivity of three peptides mixtures (BP100:RW-BP100, BP100:CA-M, and RW-BP100:CA-M), against three strains of E. amylovora representing distinct genotypes and virulence (LMG 2024, Ea 630 and Ea 680). The three AMPs’ mixtures were assayed at eight different equimolar concentrations ranging from 0.25 to 6 μM (1:1). Results showed MIC and MBC values between 2.5 and 4 μM for every AMP mixture and strain. Regarding cell viability, flow cytometry and alamarBlue reduction, showed high reduction (>25%) of viable cells after 30 min of AMP exposure, depending on the peptide mixture and strain assayed. Hypersensitive response in tobacco plants showed that the most efficient AMPs mixtures and concentrations caused low to no reaction of the plant. Altogether, the AMPs mixtures studied are better treatment solutions to control fire blight disease than the same AMPs applied individually.

The Role of Pectobacterium atrosepticum Exopolysaccharides in Plant–Pathogen Interactions

The phytopathogenic bacterium Pectobacterium atrosepticum (Pba), one of the members of the soft rot Pectobacteriaceae, forms biofilm-like structures known as bacterial emboli when colonizing the primary xylem vessels of the host plants. The initial extracellular matrix of the bacterial emboli is composed of the host plant’s pectic polysaccharides, which are gradually substituted by the Pba-produced exopolysaccharides (Pba EPS) as the bacterial emboli “mature”. No information about the properties of Pba EPS and their possible roles in Pba-plant interactions has so far been obtained. We have shown that Pba EPS possess physical properties that can promote the maintenance of the structural integrity of bacterial emboli. These polymers increase the viscosity of liquids and form large supramolecular aggregates. The formation of Pba EPS aggregates is provided (at least partly) by the acetyl groups of the Pba EPS molecules. Besides, Pba EPS scavenge reactive oxygen species (ROS), the accumulation of which is known to be associated with the formation of bacterial emboli. In addition, Pba EPS act as suppressors of the quantitative immunity of plants, repressing PAMP-induced reactions; this property is partly lost in the deacetylated form of Pba EPS. Overall, our study shows that Pba EPS play structural, protective, and immunosuppressive roles during Pba–plant interactions and thus should be considered as virulence factors of these bacteria.

Not Just a Cycle: Three gab genes Enable the Non-cyclic Flux Toward Succinate Via GABA Shunt in ‘Candidatus Liberibacter asiaticus’-infected citrus

Although the mitochondria retain all required enzymes for an intact tricarboxylic acid (TCA) cycle, plants might shift the cyclic flux from the TCA cycle to an alternative non-cyclic pathway via γ-aminobutyric acid (GABA) shunt under specific physiological conditions. We hypothesize that several genes may ease this non-cyclic flux and contribute to the citrus response to the phytopathogenic bacterium ‘Candidatus Liberibacter asiaticus’, the causal agent of Huanglongbing in citrus. To test this hypothesis, we used multi-omics techniques (metabolomics, fluxomics, and transcriptomics) to investigate the potential role(s) of putative gab homologies from Valencia sweet orange (Citrus sinensis). Our findings showed that ‘Ca. L. asiaticus’ significantly increased the endogenous GABA and succinate content but decreased ketoglutarate in infected citrus plants. Citrus genome harbors three putative gab genes including amino-acid permease (aka GABA permease; CsgabP), GABA transaminase (CsgabT), and succinate-semialdehyde dehydrogenase (aka GABA dehydrogenase; CsgabD). The transcript levels of CsgabP, CsgabT, and CsgabD were upregulated in citrus leaves upon the infection with ‘Ca. L. asiaticus’ and after the exogenous application of GABA or deuterium-labeled GABA isotope (GABA-D6). Moreover, our finding showed that exogenously applied GABA is quickly converted to succinate and fed into the TCA cycle. Likewise, the fluxomics study showed that GABA-D6 is rapidly metabolized to succinate-D4. Our work proved that GABA shunt and three predicated gab genes from citrus, support the upstream non-cyclic flux toward succinate rather than an intact TCA cycle and contribute to citrus defense responses to ‘Ca. L. asiaticus’.

Novel Nanobiocomposites Based on Natural Polysaccharides as Universal Trophic Low-Dose Micronutrients

New promising manganese-containing nanobiocomposites (NCs) based on natural polysaccharides, arabinogalactan (AG), arabinogalactan sulfate (AGS), and κ-carrageenan (κ-CG) were studied to develop novel multi-purpose trophic low-dose organomineral fertilizers. The general toxicological effects of manganese (Mn) on the vegetation of potatoes (Solanum tuberosum L.) was evaluated in this study. The essential physicochemical properties of this trace element in plant tissues, such as its elemental analysis and its spectroscopic parameters in electron paramagnetic resonance (EPR), were determined. Potato plants grown in an NC-containing medium demonstrated better biometric parameters than in the control medium, and no Mn accumulated in plant tissues. In addition, the synthesized NCs demonstrated a pronounced antibacterial effect against the phytopathogenic bacterium Clavibacter sepedonicus (Cms) and were proved to be safe for natural soil microflora.

Effect of Natural Polysaccharide Matrix-Based Selenium Nanocomposites on Phytophthora cactorum and Rhizospheric Microorganisms

We studied the effects of new chemically synthesized selenium (Se) nanocomposites (NCs) based on natural polysaccharide matrices arabinogalactan (AG), starch (ST), and kappa-carrageenan (CAR) on the viability of phytopathogen Phytophthora cactorum, rhizospheric bacteria, and potato productivity in the field experiment. Using transmission electron microscopy (TEM), it was shown that the nanocomposites contained nanoparticles varying from 20 to 180 nm in size depending on the type of NC. All three investigated NCs had a fungicidal effect even at the lowest tested concentrations of 50 µg/mL for Se/AG NC (3 µg/mL Se), 30 µg/mL for Se/ST NC (0.5 µg/mL Se), and 39 µg/mL for Se/CAR NC (1.4 µg/mL Se), including concentration of 0.000625% Se (6.25 µg/mL) in the final suspension, which was used to study Se NC effects on bacterial growth of the three common rhizospheric bacteria Acinetobacter guillouiae, Rhodococcus erythropolis and Pseudomonas oryzihabitans isolated from the rhizosphere of plants growing in the Irkutsk Region. The AG-based Se NC (Se/AG NC) and CAR-based Se NC (Se/CAR NC) exhibited the greatest inhibition of fungal growth up to 60% (at 300 µg/mL) and 49% (at 234 µg/mL), respectively. The safe use of Se NCs against phytopathogens requires them to be environmentally friendly without negative effects on rhizospheric microorganisms. The same concentration of 0.000625% Se (6.25 µg/mL) in the final suspension of all three Se NCs (which corresponds to 105.57 µg/mL for Se/AG NC, 428.08 µg/mL for Se/ST NC and 170.30 µg/mL for Se/CAR NC) was used to study their effect on bacterial growth (bactericidal, bacteriostatic, and biofilm formation effects) of the three rhizospheric bacteria. Based on our earlier studies this concentration had an antibacterial effect against the phytopathogenic bacterium Clavibacter sepedonicus that causes diseases of potato ring rot, but did not negatively affect the viability of potato plants at this concentration. In this study, using this concentration no bacteriostatic and bactericidal activity of all three Se NCs were found against Rhodococcus erythropolis based on the optical density of a bacterial suspension, agar diffusion, and intensity of biofilm formation, but Se/CAR and AG NCs inhibited the growth of Pseudomonas oryzihabitans. The cell growth was decrease by 15–30% during the entire observation period, but the stimulation of biofilm formation by this bacterium was observed for Se/CAR NC. Se/AG NC also had bacteriostatic and antibiofilm effects on the rhizospheric bacterium Acinetobacter guillouiae. There was a 2.5-fold decrease in bacterial growth and a 30% decrease in biofilm formation, but Se/CAR NC stimulated the growth of A. guillouiae. According to the results of the preliminary field test, an increase in potato productivity by an average of 30% was revealed after the pre-planting treatment of tubers by spraying them with Se/AG and Se/CAR NCs with the same concentration of Se of 0.000625% (6.25 µg/mL) in a final suspension. The obtained and previously published results on the positive effect of natural matrix-based Se NCs on plants open up prospects for further investigation of their effects on rhizosphere bacteria and resistance of cultivated plants to stress factors.

Stringent response in bacteria and plants under infection.

Stringent response (SR), a primary stress reaction in bacteria and plant chloroplasts, is a molecular switch that provides operational stress-induced reprogramming of transcription under conditions of abiotic and biotic stress. Given that the infection is a stressful situation for both partners, the host plant and the pathogen, we analyzed the expression of bacterial and plastid SR-related genes during plant-microbial interaction. In the phytopathogenic bacterium Pectobacterium atrosepticum, SpoT-dependent SR was induced after contact with potato or tobacco plants. In plants, two different scenarios of molecular events developed under bacterial infection. Plastid SR was not induced in the host plant potato Solanum tuberosum, which co-evolved with the pathogen for a long time. In this case, the salicylic acid defense pathway was activated and plants were more resistant to bacterial infection. SR was activated in the tobacco Nicotiana tabacum (experimental host) along with the activation of jasmonic acid-related genes resulting in plant death. These results are important to more fully understand the evolutionary interplay between plants and symbionts/pathogens.

Myco- and microorganisms in the tuberculosis pathology of the common ash in Ukraine and interaction between them

Attention is focused on the fact that in recent years there has been epiphytotic dieback out of many species of forest woody plants both in Ukraine and in other countries of the world, which has a dynamic character and a tendency to grow. In the deep pathology of this phenomenon, phytopathogenic bacteria, which have high reproduction energy and can penetrate the plant both from the outside and cause a pathological process as vital obligates, were left without attention. It has been established that the most common and harmful disease of common ash in Ukraine is tuberculosis. The causative agent of the disease is the phytopathogenic bacterium Pseudomonas syringae pv. savastanoi Gard. affects both trunks, branches and shoots, and inflorescences of common ash. Bacteria Pseudomonas sp., Pseudomonas fluorescens Mig., Pseudomonas syringae Van., Erwinia herbicola Eh., Xanthomonas sp. were isolated from tuberculous pathology as a concomitant myco- and microbiota and micromycetes Cladosporium cladosporiodes Fres., Ulocladium botrytis Preus., Mycelia sterilia (dark), Mycelia sterilia (orange), Fusarium heterosporum Lin., Fusarium sp., W., Cylindrocarpon didymium Har., etc. The mechanism of systemic relationships of the components of myco- and microbiota of tuberculous pathology of common ash in the regulation (self-regulation) of pathogenicity and aggressiveness of vital obligates has been investigated. Attention is focused on the prospects and expediency of using the antagonistic properties of myco- and microorganisms and biological products based on them for the prevention and protection of tree plantations from bacterial pathogens. It is shown that the pathology of common ash is a multifaceted phenomenon with interrelated processes of an infectious and non-infectious nature. The need to distinguish between the etiology and pathogenesis of this negative phenomenon is indicated, that is, not to mix the factors that lead to the weakening of ordinary ash (factors catalyzing the disease) and the factors that cause its epiphytotic dieback. Keywords: bacteria, fungi, common ash, antagonism, Pseudomonas syringae pv. savastanoi.

Expression of a Fungal Lectin in Arabidopsis Enhances Plant Growth and Resistance Toward Microbial Pathogens and a Plant-Parasitic Nematode

Coprinopsis cinerea lectin 2 (CCL2) is a fucoside-binding lectin from the basidiomycete C. cinerea that is toxic to the bacterivorous nematode Caenorhabditis elegans as well as animal-parasitic and fungivorous nematodes. We expressed CCL2 in Arabidopsis to assess its protective potential toward plant-parasitic nematodes. Our results demonstrate that expression of CCL2 enhances host resistance against the cyst nematode Heterodera schachtii. Surprisingly, CCL2-expressing plants were also more resistant to fungal pathogens including Botrytis cinerea, and the phytopathogenic bacterium Pseudomonas syringae. In addition, CCL2 expression positively affected plant growth indicating that CCL2 has the potential to improve two important agricultural parameters namely biomass production and general disease resistance. The mechanism of the CCL2-mediated enhancement of plant disease resistance depended on fucoside-binding by CCL2 as transgenic plants expressing a mutant version of CCL2 (Y92A), compromised in fucoside-binding, exhibited wild type (WT) disease susceptibility. The protective effect of CCL2 did not seem to be direct as the lectin showed no growth-inhibition toward B. cinerea in in vitro assays. We detected, however, a significantly enhanced transcriptional induction of plant defense genes in CCL2- but not CCL2-Y92A-expressing lines in response to infection with B. cinerea compared to WT plants. This study demonstrates a potential of fungal defense lectins in plant protection beyond their use as toxins.

Effects of nanoselenium as a nutrient medium component on the main cultivation parameters and antagonistic activity of Lactobacillus strains

The study aims to establish the effect of nanoselenium on the growth and antagonistic activity of the Lactobacillus casei IMB B-7343 and Lactobacillus plantarum IMB B-7344 strains against the phytopathogenic bacterium Xanthomonas campestris B-4102. Selenium nanoparticles were obtained by reducing a solution of sodium selenite with L-cysteine in the presence of sodium alginate. The cultivation of Lactobacilliwas carried out in a MRS nutrient medium with the addition of a colloidal solution of nanoselenium at the following concentrations: 0.05; 0.1; 0.15; 0.2 and 0.25 mg/l (for selenium). Bacteria were cultured in a 96-well plate in a Multiskan FC photometer at 36 ºС under constant shaking. The antagonistic activity of the strains was studied by the method of agar blocks. It was found that the addition of nanoselenium to the nutrient medium at a concentration of 0.2 to 0.25 mg/l contributed to a decrease in the duration of the exponential growth phase by an average of 3 hours, as well as to a decrease in the biomass accumulation of the L. casei IMB B-7343 strain by 15.0%. The optical density of this strain culture was observed to increase by 15.9% in the stationary growth phase under the addition of a nanoselenium solution to the nutrient medium at a concentration of 0.15 mg/l. During the cultivation of the L. plantarum IMB B-7344 strain in an MRS nutrient medium with the addition of a colloidal solution of nanoselenium at a concentration of 0.05–0.25 mg/l in the exponential development phase, an acceleration of the culture growth on average by 5 hours relative to the control was observed. It was noted that, upon the addition of nanoselenium to the nutrient medium, an increase in the antagonistic activity of lactobacillus strains against the phytopathogenic bacterium X. campestris B-4102 was observed, particularly in the L. plantarum IMB B-7344 strain (the zone of inhibition of the phytopathogen growth was 14.5–15 mm).