Ensuring the safety and efficacy of veterinary vaccines requires reliable methods for detecting microbial contamination, particularly from Mycoplasma species, which pose a significant risk in cell-culture-derived vaccines. In the Republic of Korea, polymerase chain reaction (PCR) is predominantly used for Mycoplasma testing due to its faster turnaround compared to culture-based methods. However, in combination with vaccines containing Erysipelothrix rhusiopathiae and classical swine fever virus, PCR is rendered ineffective because of cross-reactivity between Mycoplasma universal primers and E. rhusiopathiae, resulting in non-specific amplification. This limitation necessitates reliance on the labor-intensive culture method, underscoring the need for more accurate and efficient alternatives. This study aimed to develop and validate next-generation sequencing (NGS)-based methods for detecting Mycoplasma contamination in veterinary vaccines and to compare their performance with that of PCR. Five species, including Acholeplasma laidlawii (genus Acholeplasma) and four Mycoplasma species—Mycoplasma fermentans, Mycoplasma orale, Mycoplasma hyorhinis, and Mycoplasma synoviae–were spiked into samples containing E. rhusiopathiae, a common vaccine component. Two NGS-based approaches were evaluated: (1) a reference-mapping method incorporating two-step alignment and de novo assembly, and (2) a 16S rRNA-based metabarcoding analysis using DADA2 and Qiime2. The reference-mapping method effectively filtered non-specific reads and accurately reconstructed Mycoplasma-derived contigs, whereas the metabarcoding approach enabled taxonomic profiling with quantitative resolution. The detection limits of NGS-based methods were substantially lower than those of PCR, demonstrating improvements of up to 100-fold depending on the species. Notably, omission of the initial mapping step resulted in excessive non-specific contig formation, highlighting the importance of the dual-step reference-mapping strategy. Although metabarcoding provided valuable abundance data, it was more prone to non-specific hits due to limited read overlap. In conclusion, the reference-mapping method demonstrated superior sensitivity, specificity, and quantification compared to both conventional PCR and metabarcoding, supporting its use as a robust tool for vaccine quality control. Implementing NGS-based detection methods could significantly enhance the safety and effectiveness of veterinary vaccines, ultimately enhancing vaccine quality control.

Avian mycoplasmosis, caused by Mycoplasma synoviae and Mycoplasma gallisepticum, poses significant economic challenges due to respiratory issues, reduced egg production and soft eggshells. The variable lipoprotein haemagglutinin (VlhA) protein, crucial for pathogenicity, comprises conserved (MSPB) and variable (MSPA) regions. The aim of this study was to identify the conserved region of vlhA gene sequences in field strain. We examined vlhA sequences from field strains collected in central Mexico (Jalisco and Mexico City). Specifically, we analysed 124 deformed eggs and 10 laying hens from 9 farms with Hy-line and Bovans breeds. Using PCR targeting the mgc2 and 16S rRNA genes, we characterized 24 field strains, 4 of which were Myc. synoviae and 20 of which were Myc. gallisepticum. We analysed the vlhA regions, based on the AF035624.1 reference sequence, with American Type Culture Collection strains as positive controls. Additionally, we validated the PCR with 20 negative samples from Mycoplasma isolation without the need for cultivation. We identified two amplification regions: MSPB and MSPA. Bioanalysis revealed relationships between our field samples and avian Mycoplasma sequences in GenBank, alongside similarities with lipoproteins present in Acholeplasma laidlawii PG8 and Escherichia coli. Given the significance of the VlhA protein in pathogenicity and immune evasion, the identified conserved sequences hold potential as therapeutic targets and for phylogenetic studies.

Bovine mycoplasma mastitis is highly transmittable and hard to treat by chemotherapy. It causes severe economic loss and is considered a major problem for milk production. Mycoplasma canadense is one of the causative agents of mycoplasma mastitis. A primer set to detect M. canadense was developed based on single nucleotide polymorphism-specific loop-mediated isothermal amplification. Using this primer set, 10 fg M. canadense DNA corresponding to the DNA amount of ~13 cells was detected within 40 min. Cross-reactivity with other bovine Mycoplasma spp., Acholeplasma laidlawii, and mastitis-related bacteria was not observed when ≤1 pg DNA was applied. These results would provide a basis for validating future experiments with spiked-milk and field samples for the development of rapid detection of M. canadense.

Carotenoids are a class of highly hydrophobic compounds synthesized by plants in limited quantities. This study explores the potential for increasing the production yield of lycopene, a typical carotenoid compound, through engineered Escherichia coli. Given that lycopene biosynthesis occurs within microbial hosts and it is subsequently stored within lipid membranes, this study focuses on the impact of inducing membrane vesicles on lycopene yield by expressing monoglycosyldiacylglycerol synthase (MGS) or diglucosyldiacylglycerol synthase (DGS) from Acholeplasma laidlawii and inserting the upstream isopentenol utilization pathway (IUP) into the chromosome. The effect of MGS and DGS on lipid production in the cell was quantified. The results show that inserting the IUP into the chromosome increased the specific lycopene yield by 2.1-fold compared to the plasmid-based system when using a PproD constitutive promoter and by 2.0-fold when using the inducible Ptrc promoter. The expression of MGS and DGS resulted in a small increase of 31% and 33% (w/w) lipid content, respectively. When expressed in lycopene producing strains, the lycopene content decreased in the IUP strains but increased in the negative control strain expressing only the native MEP pathway from undetectable levels to 0.34 ± 0.08 mg/g.

Gene- and genome-based approaches were used to determine whether Vigna little leaf (ViLL) phytoplasma, which occurs in northern Australia, is a distinct ‘Candidatus Phytoplasma’ species. The ViLL 16S rRNA gene sequences exhibited the highest known similarity to species in the 16SrXXIX-A and 16SrIX-D subgroups, namely ‘Candidatus Phytoplasma omanense’ (98.03–98.10%) and ‘Candidatus Phytoplasma phoenicium’ (96.87–97.20%), respectively. A total of 48 single-copy orthologue genes were identified to be shared among the two draft ViLL phytoplasma genomes, 30 publicly available phytoplasma genomes, and one Acholeplasma laidlawii genome as the outgroup taxon. Phylogenomic assessments using the 48 shared single-copy orthologue genes supported that ViLL and ‘Ca. Phytoplasma phoenicium’ were closely related yet distinct species. The 16S rRNA gene sequence analysis and phylogenomic assessment indicate that ViLL phytoplasmas are a distinct taxon. As such, a novel species, ‘Candidatus Phytoplasma vignae’, is proposed. Strain BAWM-336 (genome accession number JAUZLI000000000) detected in Momordica charantia (bitter melon) serves as the reference strain of this species, with infected plant material deposited in the Victorian Plant Pathology Herbarium (VPRI) as VPRI 44369.

We have developedgenetic tools for the atypical bacterium Acholeplasma laidlawii. A. laidlawii isa member of the class Mollicutes, which lacks cellwalls, has small genomes, and has limited metabolic capabilities,requiring many metabolites from their hosts. Several of these traitshave facilitated the development of genome transplantation for some Mollicutes, consequently enabling the generation of syntheticcells. Here, we propose the development of genome transplantationfor A. laidlawii. We first investigated a donor–recipientrelationship between two strains, PG-8A and PG-8195, through whole-genomesequencing. We then created multihost shuttle plasmids and used themto optimize an electroporation protocol. We also evolved a superiorstrain for DNA uptake via electroporation. We created a PG-8A donorstrain with a Tn5 transposon carrying a tetracycline resistance gene.These tools will enhance Acholeplasma research andaccelerate the effort toward creating A. laidlawii strains with synthetic genomes.

Small heat shock proteins (sHSPs) represent a first line of stress defense in many bacteria. The primary function of these molecular chaperones involves preventing irreversible protein denaturation and aggregation. In Escherichia coli, fibrillar EcIbpA binds unfolded proteins and keeps them in a folding-competent state. Further, its structural homologue EcIbpB induces the transition of EcIbpA to globules, thereby facilitating the substrate transfer to the HSP70-HSP100 system for refolding. The phytopathogenic Acholeplasma laidlawii possesses only a single sHSP, AlIbpA. Here, we demonstrate non-trivial features of the function and regulation of the chaperone-like activity of AlIbpA according to its interaction with other components of the mycoplasma multi-chaperone network. Our results show that the efficiency of the A. laidlawii multi-chaperone system is driven with the ability of AlIbpA to form both globular and fibrillar structures, thus combining functions of both IbpA and IbpB when transferring the substrate proteins to the HSP70-HSP100 system. In contrast to EcIbpA and EcIbpB, AlIbpA appears as an sHSP, in which the competition between the N- and C-terminal domains regulates the shift of the protein quaternary structure between a fibrillar and globular form, thus representing a molecular mechanism of its functional regulation. While the C-terminus of AlIbpA is responsible for fibrils formation and substrate capture, the N-terminus seems to have a similar function to EcIbpB through facilitating further substrate protein disaggregation using HSP70. Moreover, our results indicate that prior to the final disaggregation process, AlIbpA can directly transfer the substrate to HSP100, thereby representing an alternative mechanism in the HSP interaction network.

Rejection and fouling of track-etched microfiltration membranes by Acholeplasma laidlawii: Clues to mycoplasma behavior during “sterile” dead-end filtration

The division protein FtsZ interacts with the small heat shock protein IbpA in Acholeplasma laidlawii

This work describes curious structures formed by the mainly phytopathogenic mycoplasma Acholeplasma laidlawii, as well as the human pathogen Ureaplasma parvum cells which resemble cell-in-cell structures of higher eukaryotes and protists. The probable significance of such structures for the mycoplasma cell is discussed. The possibility of their formation in nature and their potential role in the transformation of genetic material, for example, by maintaining (on the one hand) the stability of the genome in the line of generations during asexual reproduction or (on the other hand) the genome plasticity, are substantiated. It should be especially noted that all the arguments presented are based only on morphological data. However, closer attention to unusual structures, the existence of which was shown by electron microscopy images in this case, may prompt researchers to analyze their data more carefully and find something rare and non-trivial among seemingly trivial things. If it is proven by additional methods that cell-in-cell structures can indeed be formed by prokaryotes without a cell wall, this phenomenon may acquire general biological significance.

The considerable utility of glycoside phosphorylases (GPs) has led to substantial efforts over the past two decades to expand the breadth of known GP activities. Driven largely by the increase of available genomic DNA sequence data, the gap between the number of sequences in the carbohydrate active enzyme database (CAZy DB) and its functionally characterized members continues to grow. This wealth of sequence data presented an exciting opportunity to explore the ever-expanding CAZy DB to discover new GPs with never-before-described functionalities. Utilizing an in silico sequence analysis of CAZy family GH94, we discovered and then functionally and structurally characterized the new GP β-1,3-N-acetylglucosaminide phosphorylase. This new GP was sourced from the genome of the cell-wall-less Mollicute bacterium, Acholeplasma laidlawii and was found to synthesize β-1,3-linked N-acetylglucosaminide linkages. The resulting poly-β-1,3-N-acetylglucosamine represents a new, previously undescribed biopolymer that completes the set of possible β-linked GlcNAc homopolysaccharides together with chitin (β-1,4) and PNAG (poly-β-1,6-N-acetylglucosamine). The new biopolymer was denoted acholetin, a combination of the genus Acholeplasma and the polysaccharide chitin, and the new GP was thus denoted acholetin phosphorylase (AchP). Use of the reverse phosphorolysis action of AchP provides an efficient method to enzymatically synthesize acholetin, which is a new biodegradable polymeric material.

Acholeplasma (A.) laidlawii is an opportunistic pathogen with the ability to disseminate resistance determinants to antibiotics; however, its resistance to macrolides has been less studied. The aim of the present study was to characterize the mechanisms responsible for the resistance to macrolides, tiamulin and lincomycin found in a strain of A. laidlawii isolated from a pig with pneumonia. MICs of erythromycin, 15- and 16-membered macrolides, tiamulin and lincomycin were determined by microdilution method with and without reserpine, an inhibitor of ABC efflux pumps and regions of the genome were sequenced. Reserpine only decreased lincomycin MIC but it did not change the MICs of macrolides and tiamulin. The analysis of the DNA sequence of 23S rRNA showed nucleotide substitutions at eight different positions, although none of them were at positions previously related to macrolide resistance. Five mutations were found in the L22 protein, one of them at the stop codon. In addition, two mutations were found in the amino acid sequence of L4. The combination of multiple mutations in the ribosomal proteins L22 and L4 together with substitutions in 23S rRNA DNA sequence was associated with the resistance to macrolides, the pleuromutilin and lincomycin in the studied A. laidlawii strain.

For the first time it was shown that the development of resistance to ciprofloxacin in vitro in Acholeplasma laidlawii, a mycoplasma which is widely spread in nature and which is the main contaminant of cell cultures and vaccines, is associated with diverse pathways of virulence evolution: virulome and virulence differ significantly between ciprofloxacin-resistant strains, including those with the same level of antimicrobial resistance.

It is known that the ratio of the main groups of microorganisms in the microbiocenosis form the biological fertility of the soil. In this regard, it is necessary to take into account the influence of biologically active substances, including nanoparticles and their derivatives, used in crop production, on the composition of soil microbiota and its biodiversity. The aim of this study was to investigate the effect of Galega orientalis plants inoculation with phytopathogenic microorganisms and foliar treatment of plants with nanochelates on the total amount of microbial groups in rhizosphere of Galega orientalis. Methods. Acholeplasma laidlawii var. granulum 118 UCM BM–34 was cultivated in the liquid nutrient medium CM IMV-72 (pH 7.8) in thermostat at 32°C for 72 hours. Pseudomonas syringae pv. atrofaciens D13 was cultivated on potato agar in thermostat at 26–28°C. For artificial infection a bacterial suspension with a concentration of 1×109 CFU/mL according to the turbidity standard was prepared. Galega orientalis plants were inoculated with phytopathogenic strains of microorganisms by subepidermal injection into the stem. The total count of microbial groups in the samples was performed by the method of plating on selective media, the result was expressed in colony-forming units (CFU). For statistical processing of data, calculations of the arithmetic mean and its standard error were performed. Results. It was found that the total amount of microorganisms, the number of actinomycetes and oligotrophic microorganisms increased and the number of micromycetes decreased in the rhizosphere of Galega orientalis plants infected with phytoplasma. In contrast to phytoplasma infected plants, the number of aerobic nitrogenfixing bacteria in the rhizosphere increased in plants infected with P. syringae pv. atrofaciens D13. Foliar treatment of Galega orientalis plants with nanochelate solutions had varying influence on the composition of microbial groups. The total amount of aerobic nitrogen-fixing bacteria increased after foliar treatment with nanochelates in the next order: V˃Ge˃Se, and the total amount of actinomycetes increased after foliar treatment in the next order: Se˃V˃Ge. The total amount of micromycetes increased in the rhizosphere of Galega orientalis after foliar treatment with Ge and Se nanochelates. It should be noted that the most significant increase in the number of oligotrophic microorganisms was observed in the rhizosphere of plants after I-Se foliar treatment. Conclusions. The number of actinomycetes, micromycetes and oligotrophic microorganisms in the rhizosphere of Galega orientalis plants infected with A. laidlawii and P. syringae increased compared to control plants; this process was associated with changes in the chemical composition of root secretions, probably due to reducing of carbohydrates and the presence of stress signal molecules. Plants infection with these pathogens had different effect on the total amount of aerobic nitrogen-fixing bacteria, the number of which increased in the rhizosphere after infection with P. syringae and decreased after infection with A. laidlawii, which may be due to the different chemical composition of the root secretions. Foliar treatment with Se, Ge and V nanochelates had the most favorable influence on the soil microbiota, causing an increase in the total amount of microbial groups, including species capable of disease development inhibiting and participating in the nutrient cycle (aerobic nitrogen-fixing bacteria, actinomycetes, micromycetes). The foliar treatment of plants with I-Se nanochelates can be considered as conditionally favorable due to a significant increase in the number of oligotrophic microorganisms, which indicates the depletion of readily available nutrients in the soil. However, the improvement of root system growth at I-Se foliar treatment of plants affected by both phytoplasma and bacterial pathogen may be a sign of stimulation of the absorptive capacity of roots, which requires further research.

Contagious bovine mastitis caused by Mycoplasma bovis and other Mycoplasma species including Mycoplasma californicum, Mycoplasma bovigenitalium, Mycoplasma alkalescens, Mycoplasma arginini, and Mycoplasma canadense is an economical obstacle affecting many dairy herds throughout California and elsewhere. Routine bacteriological culture-based assays for the pathogens are slow and subject to false-positive results due to the presence of the related, non-pathogenic species Acholeplasma laidlawii. To address the need for rapid and accurate detection methods, a new TaqMan multiplex, quantitative real-time PCR (qPCR) assay was developed that targets the 16S rRNA gene of Mycoplasma, rpoB gene of M. bovis, and the 16S to 23S rRNA intergenic transcribed spacer (ITS) region of A. laidlawii. qPCR amplification efficiency and range of detection were similar for individual assays in multiplex as when performed separately. The multiplex assay was able to distinguish between M. bovis and A. laidlawii as well as detect Mycoplasma spp. collectively, including Mycoplasma californicum, Mycoplasma bovigenitalium, Mycoplasma canadense, Mycoplasma arginini and Mycoplasma alkalescens. In milk, the lower limit of detection of M. bovis, M. californicum, and A. laidlawii with the multiplex assay was between 120 to 250 colony forming units (CFU) per mL. The assay was also able to simultaneously detect both M. bovis and A. laidlawii in milk when present in moderate (103 to 104 CFU/mL) to high (106 to 107 CFU/mL) quantities. Compared to laboratory culture-based methods, the multiplex qPCR diagnostic specificity (Sp) was 100% (95% CI [86.8–100]; n = 26) and diagnostic sensitivity (Se) was 92.3% (95% CI [74.9–99.1]; n = 26) for Mycoplasma species in milk samples collected from California dairy farms. Similarly, the Sp was 100% (95% CI [90.5–100]; n = 37) and Se was 93.3% (95% CI [68.1–99.8]; n = 15) for M. bovis. Our assay can detect and distinguish among M. bovis, other prevalent Mycoplasma spp., and non-pathogenic Acholeplasma laidlawii for effective identification and control of mycoplasma mastitis, ultimately supporting dairy cattle health and high-quality dairy products in California.

Transmission electron microscopy of cell sample sections is a popular technique in microbiology. Currently, ultrathin sectioning is done on resin-embedded cell pellets, which consumes milli- to deciliters of culture and results in sections of randomly orientated cells. This is problematic for rod-shaped bacteria and often precludes large-scale quantification of morphological phenotypes due to the lack of sufficient numbers of longitudinally cut cells. Here we report a flat embedding method that enables observation of thousands of longitudinally cut cells per single section and only requires microliter culture volumes. We successfully applied this technique to Bacillus subtilis, Escherichia coli, Mycobacterium bovis, and Acholeplasma laidlawii. To assess the potential of the technique to quantify morphological phenotypes, we monitored antibiotic-induced changes in B. subtilis cells. Surprisingly, we found that the ribosome inhibitor tetracycline causes membrane deformations. Further investigations showed that tetracycline disturbs membrane organization and localization of the peripheral membrane proteins MinD, MinC, and MreB. These observations are not the result of ribosome inhibition but constitute a secondary antibacterial activity of tetracycline that so far has defied discovery.

The extracellular vesicles (EVs) produced by bacteria transport a wide range of compounds, including proteins, DNA and RNA, mediate intercellular interactions, and may be important participants in the mechanisms underlying the persistence of infectious agents. This study focuses on testing the hypothesis that the EVs of mycoplasmas, the smallest prokaryotes capable of independent reproduction, combined in the class referred to as Mollicutes, can penetrate into eukaryotic cells and modulate their immunoreactivity. To verify this hypothesis, for the first time, studies of in vitro interaction between human skin fibroblasts and vesicles isolated from Acholeplasma laidlawii (the ubiquitous mycoplasma that infects higher eukaryotes and is the main contaminant of cell cultures and vaccines) were conducted using confocal laser scanning microscopy and proteome profiling, employing a combination of 2D-DIGE and MALDI-TOF/TOF, the Mascot mass-spectrum analysis software and the DAVID functional annotation tool. These studies have revealed for the first time that the extracellular vesicles of A. laidlawii can penetrate into eukaryotic cells in vitro and modulate the expression of cellular proteins. The molecular mechanisms behind the interaction of mycoplasma vesicles with eukaryotic cells and the contribution of the respective nanostructures to the molecular machinery of cellular permissiveness still remain to be elucidated. The study of these aspects is relevant both for fundamental research into the "logic of life" of the simplest prokaryotes, and the practical development of efficient control over hypermutable bacteria infecting humans, animals and plants, as well as contaminating cell cultures and vaccines.

The current study aimed to detect microbial causes of she camel’s subclinical and clinical mastitis and antibiotic sensitivity test for some bacterial isolates. A total of 196 milk samples were collected from mastitic and apparent healthy she camels, milk samples of apparent healthy she camels were examined by California mastitis test for detection of subclinical mastitis. All samples were cultivated on different media for detection of bacteria and fungi causing mastitis. S. aureus and Acholeplasma were subjected to antibiotic sensitivity test to detect antibiotic of choice. Out of 40 apparently healthy she camels, subclinical mastitis was detected in 19 (47.5%). Out of 196 she camels milk samples, 40 samples were positive for bacterial isolation (20.4%). E. coliandStaphylococcus epidermidis were the most predominant isolated bacteria from apparently healthy she camel milk samples while Pseudomonas aeruginosa, Staphylococcus epidermidis and Streptococcus agalactia from mastitic she camel's milk samples. This is the first record for isolation of Acholeplasma laidlawii in South Sinai where 4 Acholeplasma laidlawii isolates were isolated from mastitic and apparent healthy she camel milk samples. Aspergillus niger was the most predominant fungi followed by Candida albicans. The most predominant mixed bacterial infection in apparent healthy she camels wasProteus vulgaris and Staphylococcus epidermidis while from mastitic she camels wasPseudomonas aeruginosa and Klebsiella pneumoniae. S. aureus was sensitive to gentamycin, streptomycin, erythromycin, tetracycline, ciprofloxacin and nitrofurantion. Acholeplasma laidlawii was sensitive to gentamycin. From the obtained results, it was concluded that she camels’ mastitis was caused by several bacteria and fungi either by single or mixed infection.

The presence of a gene encoding small heat shock protein IbpA in Acholeplasma laidlawii (AlIbpA) appears to be one of the key factors determining the high adaptive capabilities of this mycoplasma. Previously, we showed a participation of the N-and C-terminal regions of AlIbpA in functions of chaperone in vitro. The aim of this work was to establish the involvement of the N - and C-terminal motifs of the recombinant AlIbpA in the survival of Escherichia coli cells under temperature stress in vivo. To determine this, we used genetically engineered versions of the AlIbpA with truncations and mutations in the N - and C-terminal domains. Viability was determined by counting CFU and differential fluorescent staining. Studies have shown that hyperproduction of the N-termini-modified IbpA from AlIbpA is able to exhibit thermotolerance E. coli cells.This research was funded by the Russian Science Foundation (project No. 17-74-20065), the microscopy assays were funded by RFBR (project № 20-34-90066).

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.