Lateral flow immunoassay (LFIA) is a convenient tool for rapid field-based control of various bacterial targets. However, for many applications, the detection limits obtained by LFIA are not sufficient. In this paper, we propose enlarging gold nanoparticles’ (GNPs) size to develop a sensitive lateral flow immunoassay to detect Ralstonia solanacearum. This bacterium is a quarantine organism that causes potato brown rot. We fabricated lateral flow test strips using gold nanoparticles (17.4 ± 1.0 nm) as a label and their conjugates with antibodies specific to R. solanacearum. We proposed a signal enhancement in the test strips’ test zone due to the tetrachloroauric (III) anion reduction on the GNP surface, and the increase in size of the gold nanoparticles on the test strips was approximately up to 100 nm, as confirmed by scanning electron microscopy. Overall, the gold enhancement approach decreased the detection limit of R. solanacearum by 33 times, to as low as 3 × 10⁴ cells∙mL^–1 in the potato tuber extract. The achieved detection limit allows the diagnosis of latent infection in potato tubers. The developed approach based on gold enhancement does not complicate analyses and requires only 3 min. The developed assay together with the sample preparation and gold enlargement requires 15 min. Thus, the developed approach is promising for the development of lateral flow test strips and their subsequent introduction into diagnostic practice.

Isothermal amplifications allow for the highly sensitive detection of nucleic acids, bypassing the use of instrumental thermal cycling. This work aimed to carry out an experimental comparison of the four most promising techniques: recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) coupled with lateral flow test or coupled with additional amplification based on CRISPR/Cas12a resulting from the fluorescence of the Cas12a-cleaved probe. To compare the four amplification techniques, we chose the bacterial phytopathogen Erwinia amylovora (causative agent of fire blight), which has a quarantine significance in many countries and possesses a serious threat to agriculture. Three genes were chosen as the targets and primers were selected for each one (two for RPA and six for LAMP). They were functionalized by labels (biotin, fluorescein) at the 5′ ends for amplicons recognition by LFT. As a result, we developed LAMP-LFT, LAMP-CRISPR/Cas, RPA-LFT, and RPA-CRISPR/Cas for E. amylovora detection. The detection limit was 10⁴ CFU/mL for LAMP-LFT, 10³ CFU/mL for LAMP-CRISPR/Cas, and 10² CFU/mL for RPA-LFT and RPA-CRISPR/Cas. The results of four developed test systems were verified by qPCR on a panel of real samples. The developed assays based on RPA, LAMP, CRISPR/Cas12a, and LFT are rapid (30–55 min), user-friendly, and highly sensitive for E. amylovora detection. All proposed detection methods can be applied to fire blight diagnosis and effective management of this disease.

The detection limit of lateral flow immunoassay (LFIA) is largely determined by the properties of the label used. We compared four nanoparticle labels differing in their chemical composition and colour: (1) gold nanoparticles (Au NPs), red; (2) Au-core/Pt-shell nanoparticles (Au@Pt NPs), black; (3) latex nanoparticles (LPs), green; and (4) magnetic nanoparticles (MPs), brown. The comparison was carried out using one target analyte—Erwinia amylovora, the causal bacterial agent of fire blight. All nanoparticles were conjugated with antibodies through methods that provide maximum functional coverage like physical adsorption (Au NPs, Au@Pt NPs) and covalent bonding (LPs, MPs). All conjugates demonstrated the same ability to bind with E. amylovora through enzyme-linked immunosorbent assay where optical properties of the nanoparticles do not determine the registered signal. However, half-maximal binding was achieved at different numbers of nanoparticles because they differ in size. All conjugates based on four nanoparticle labels were used for lateral flow assays. As a result, Au@Pt NPs provided the minimal detection limit that corresponded to 10³ CFU/mL. Au NPs and LPs detected 10⁴ CFU/mL, and MPs detected 10⁵ CFU/mL. The results highlight that simply choosing a coloured label can significantly affect the detection limit of LFIA.

Bacterial communities associated with medicinal plants are an essential part of ecosystems. The rhizosphere effect is rather important in the cultivation process. The purpose of the study was to analyze the rhizosphere effect of oregano (Origanum vulgare L.), peppermint (Mentha piperita L.), thyme (Thymus vulgaris L.), creeping thyme (Thymus serpillum L.) and sage (Salvia officinalis L.). To estimate the quantity of 16S bacteria ribosomal genes, qPCR assays were used. To compare bacterial communities’ structure of medicinal plants rhizosphere with bulk soil high-throughput sequencing of the 16S rRNA targeting variable regions V3–V4 of bacteria was carried out. The highest bacterial abundance was associated with T. vulgaris L., M. piperita L. and S. officinalis L., and the lowest was associated with the O. vulgare L. rhizosphere. Phylum Actinobacteriota was predominant in all rhizosphere samples. The maximum bacterial α-diversity was found in S. officinalis L. rhizosphere. According to bacterial β-diversity calculated by the Bray–Curtis metric, T. vulgaris L. root zone significantly differed from bulk soil. The rhizosphere effect was positive to the Myxococcota, Bacteroidota, Verrucomicrobiota, Proteobacteria and Gemmatimonadota.

In the last two decades, fire blight has progressively spread eastward from Europe and the Mediterranean area to several pome-fruit producing regions of Asia. Its causative agent, the bacterial pathogen Erwinia amylovora, was detected in several new countries, including Georgia, Kyrgyzstan, and Kazakhstan. In the latter two states, the disease creates a threat not only to the commercial apple and pear production, but also to the wild Malus and Pyrus species that constitute the basis of the local forest ecosystems. In this study, we investigated the genetic diversity of the pathogen in Central Asia and the Caucasus region utilizing CRISPR Repeat Regions (CRRs) genotyping and genome sequencing, with the aim to understand its dissemination patterns across the continent. Genome sequence analysis revealed that all strains from these two regions exclusively derived from the archetypal CRR1 genotype A. Our analysis revealed three main E. amylovora clades in Central Asia, with distinct yet partial overlapping geographical distributions. Genomic relationships among isolates indicate that Central Asian strains are genetically closest to those from the Persian region and the Middle East, while the Georgian population is genetically more distant and can align with strains from the Volga District in southern Russia and the Eastern Mediterranean area. Notably, this study also includes strains from the first confirmed occurrences of fire blight in Uzbekistan, Tajikistan, and China. Our findings highlight the importance of phylogenetic analysis and genome sequencing in understanding the phytopathogen epidemics and protecting key agricultural species and the genetic resources of their wild counterparts in the forest.

Background. Clonal apple rootstocks are one of the main components of intensive gardening. The degree of rootstock damage by fire blight affects the resistance of the variety–rootstock combination. The paper presents a study on marking quantitative trait loci (QTL) of resistance to fire blight Erwinia amylovora in clonal apple rootstock.Materials and methods. A collection of 20 rootstock forms was analyzed. For the study, SCAR markers GE-8019, AE10-375 and microsatellite marker CH-F7-FB1 were used.Results. Polymorphism was observed for all three markers, and their various combinations in one genotype were revealed. It was previously noted that genotypes that carry all three markers were more resistant than those that lack them. The presence of all three markers was observed only in forms 62-396 (В10), 16-1 and 2-9-102. The other genotypes did not have the GE8019 marker. The AE10-375 marker was identified in eight clonal rootstocks. Microsatellite marker CH-F7-FB1 was present in all tested rootstocks. However, polymorphism was detected there. Most genotypes had a 174 bp fragment, but a 210 bp fragment was identified in two of the 20 forms. Clonal rootstock 70-20-21 proved heterozygous for this marker. The analyzed collection also included samples that had only the microsatellite marker: G16, Malysh Budagovskogo, Paradizka Budagovskogo (B9), 54-118 (В118), 57-491, 70-20-20 (В119), 70-20-21, 71-7-22, 76-3-6, 83-1-15, 87-7-12, and 2-12-10. The study of rootstock forms on the basis of resistance to metabolites of the fire blight pathogen was carried out under laboratory conditions using the E. amylovora culture filtrate in vitro on leaf explants. Most of the studied genotypes had different combinations of markers. However, the experiments showed that forms 62-396 and 14-1 with two out of three markers (AE10-375 and CH-F7-FB1) phenotypically manifested the trait of resistance to metabolites of E. amylovora.