Characterization of antimicrobial substances from natural isolates of Bacillus sp. for application in biological control of plant pathogenic bacteria and fungi

Bolesti biljaka izazvane infekcijama patogenim bakterijama i gljivama mogu dovesti do smanjenja kapaciteta biljnog rasta ili naneti mnogo ozbiljniju štetu dovodeći do smrti biljaka i značajnih gubitaka u proizvodnji hrane. Veliki broj istraživanja je u poslednje vreme posvećen proučavanju roda Bacillus kao producenta sekundarnih metabolita u kontroli različitih biljnih patogena. Analizom diverziteta gena odgovornih za produkciju neribozomalno sintetisanih lipopeptida kolekcije od 205 Bacillus sp. izolata utvrđeno je da je za najveći broj izolata (171) ustanovljen DNK fragment očekivane dužine, koji odgovara bacilomicin D sintetazi, dok su u sličnom broju izolata dobijeni fragmenti očekivanih dužina za kurstakinske (154) i surfaktinske operone (142), a prisustvo iturinskog operona ustanovljeno je kod 84, odnosno 59 izolata u slučaju gena za fengicin sintetazu. Prisustvo svih pet biosintetičkih operona je potvrđeno kod 11 izolata. Najveća produkcija antimikrobnih jedinjenja za pet izabranih izolata je pokazana u eksponencijalnoj ili ranoj stacionarnoj fazi rasta. Prikazan je jak antifungalni efekat lipopeptidnih ekstrakata prema više od 30 testiranih gljiva, sa najnižim zabeleženim minimalnim inhibitornim koncentracijama od 0,008 mg/ml protiv Fusarium semitectum, kao i izražen antibakterijski potencijal, naročito protiv fitopatogenih vrsta rodova Xanthomonas i Pseudomonas, sa najnižom minimalnom inhibitornom koncentracijom od 0,06 mg/ml. Proučavanje interakcija pojedinačnih lipopeptidnih ekstrakata u međusobnim, kao i u kombinacijama sa etarskim uljima, ukazalo je na postojanje sinergističkog efekta za neke kombinacije u in vitro, odnosno aditivnog efekta u in situ uslovima, na bakterijskim i fungalnim patogenima. Utvrđivanjem direktnog antagonističkog efekta lipopeptidnih jedinjenja in planta, ustanovljena je značajna redukcija nekroze tkiva listova Arabidopsis thaliana L. i Beta vulgaris L., tretiranih bakterijskim patogenima. Takođe, u in situ uslovima postignuta je značajna redukcija nekroze tkiva ploda jabuke izazvana fungalnom infekcijom...Plant diseases caused by infection with pathogenic bacteria and fungi can lead to the reduction in the capacity of plant growth or can cause far more serious damage, leading to the death of plants and significant losses in food production. Numerous recent studies are devoted to the genus Bacillus as a producer of secondary metabolites in the control of different plant pathogens. In the analysis of diversity of genes responsible for the nonribosomal synthesized lipopeptides in collections of 205 Bacillus sp. isolates, for the largest number of isolates (171) the DNA PCR fragment of the expected length, corresponding to bacillomycin D synthases was obtained, while in a similar number of isolates fragments of the expected length for kurstakin (154) and surfactin operons (142) were obtained. The presence of iturin operon in the 84 or 59 isolates in the case of the gene for fengycin synthetase, was also found. The presence of all five biosynthetic operons in the 11 isolates was confirmed as well. The maximum production of antimicrobial compounds for five selected isolates in exponential or early stationary phase of growth was determined. Strong antifungal effect of lipopeptide extracts towards more than 30 fungi tested, with lowest recorded minimal inhibitory concentrations of 0,008 mg/ml against Fusarium semitectum, as well as expressed antibacterial potential, in particular against phytopathogenic species of the genera Pseudomonas and Xanthomonas, with the lowest minimum inhibitory concentration of 0,06 mg/ml was determined. In the analysis of the interaction of individual lipopeptide extracts mutually, as well as in combination with essential oils, the existence of a synergistic effect in vitro respectively additive effect in situ, on bacterial and fungal pathogens, for some combinations were shown. Establishing a direct antagonistic effect of lipopeptide compounds in planta, significant reduction in necrotic lesions of leaves of Arabidopsis thaliana L. and Beta vulgaris L. treated with bacterial pathogens was obtained. Also, a significant reduction in necrotic lesions of apple fruit caused by fungal infection in situ was determined..

BIOCTA: Novel approach to biocontrol of recently described plant tumorogenic Rhizobium spp. using autochthonous microbial solutions

Introduction: A novel group of Rhizobiumspp. strains belonging to the “tumorigenes” clade has recently been described on blackberry in Serbia and Germany and on rhododendron in Germany. The BIOCTA project aimed to characterize efficient plant-associated bacterial strains for biocontrol of crown gall, thus providing an environmentally friendly alternative to pesticides that would contribute to the development of sustainable agriculture. Methods: Antagonistic potential of 37 biocontrol strains against two R. tumorigenes strains 932 and 1078 and Rhizobium sp. strain rho-6.2 was evaluated in vitro using the “well diffusion” method, as well as in vivo on tomato plants, using two inoculation strategies (co-inoculation and preventive). DNA metabarcoding approach was used to analyze the phytobiome of treated and non-treated tomato plants. Results: Based on the determined in vitro antagonistic potential, seven strains – Bacillus spp. (B. amyloliquefaciens ID084 and GT28.3, B. velezensis X5-2, and B. subtilis GD1), Pseudomonas sp. (R-6.10 and R11-20) and Agrobacterium rosae rho-6.1 were selected for further in vivo experiments. Of all tested strains/treatments, two Pseudomonas strains were the most efficient, showing up to 92.86%efficacy in suppressing tumors caused by Rhizobiumsp. strain rho-6.2 when applied in a co-inoculation strategy. Based on the DNA metabarcoding analysis, genera Pseudolabrys and Asanoa prevailed in the co-inoculation strategy, while Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium prevailed in positive control. Conclusion: Crown gall tumors have shown to be a valuable source of antagonistic isolates. Pseudomonas strains R-6.10 and R11-20 could be proposed for the efferent control of crown gall caused by newly described Rhizobium spp. strains in nurseries

PSEUDOMONAS SPP. IN BIOCONTROL OF CROWN GALL DISEASE: NEW APPROACHES

INTRODUCTION Crown gall is an economically important and widespread plant disease caused by tumorigenic bacteria that are commonly affiliated with the genera Agrobacterium, Allorhizobium and Rhizobium. Novel and an atypical group of tumorigenic agrobacteria belonging to the genus Rhizobium (“tumorigenes” clade) was identified as a causative agent of crown/cane gall on blackberry, rhododendron and blueberry in Serbia and Germany (Kuzmanović et al., 2018 and 2019). Efficient measures to control crown gall disease were not reported till nowadays, so assessment and application of alternative biological control measures would contribute to sustainable agricultural production and environmental protection. The aims of the study were 1) identification of candidate bacterial strains that could be employed for biological control 2) to analyse phytobiome of the treated and non-treated crops and 3) to perform a whole-genome sequencing of a few most promising biocontrol strains. STATE OF THE ART Antimicrobial activity of ten biocontrol candidates from rhododendron and 27 additional antagonistic strains were tested in vitro against the tumor-inducing strain Rhizobium sp. rho-6.2. The six most efficient Pseudomonas and Bacillus strains were tested in vivo, using co-inoculation and preventive inoculation strategies in controlled greenhouse conditions on tomato plants as a model system in four replicas and randomized. Tumors from the most effective treatments were sampled, and then total DNA was isolated and subjected to the next-generation sequencing (NGS). Direct analysis of bacterial communities using Illumina MiSeq sequencing of 16S rRNA gene amplicon libraries was performed to assess the microbial ecological effect, with complete bioinformatic and computational biology analysis conducted. Also, a whole-genome sequencing of a few most promising antagonistic strains was performed. RESULTS Among six antagonistic strains, the most efficient in co-inoculation strategy against pathogenic Rhizobium sp. rho-6.2 were two Pseudomonas strains (R-6.10 and R-11.20), which reduced a tumor size 92.86%. The same Pseudomonas strains were less effective in preventive treatments (15.38 and 30.77%). Although Bacillus strains exhibited high in vitro antimicrobial activity, their in vivo activity was in preventive treatment only 15.38%, whilst in co-inoculation strategy was detected as moderate (42.86%). Bacillus and Pseudomonas strains applied together increased biocontrol activity with 38.6% of tumor’s reduction. In analyzed treatments, was detected the dominant presence of Proteobacteria followed by a moderate presence of Actinobacteriota and Firmicutes. On the genus level, the most abundant, both in negative control and treatments, were representatives of Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium group (18,53% - 71,81%) followed by Pseudomonas spp. (2,76%- 36,46%). According to alpha diversity indexes on the genus level, the highest values were detected in the negative control, pre-treatment with Pseudomonas sp. R-6.10, co-inoculation with Pseudomonas spp. R-6.10 and R-11.20 individually. Analysis of beta diversity by the DPCoA matrix exhibited that the co-inoculation and positive control groups were well separated, whilst preventive treatment overlapped both the co-inoculation and positive control samples. Differential abundance analysis on a genus level revealed a statistically higher presence of Stenotrophomonas and Asanoa in preventive treatments and Dyadobacter and Pandoraea spp. in their positive control. In the co-inoculation strategy, Pseudolabrys and Asanoa were prevalent in treatments and Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium was detected as prevalent in positive control. Whole-genome sequencing and preliminary comparative genomics analyses revealed that the best biocontrol candidates, Pseudomonas strains R-6.10 and R-11.20 represent two new species, most closely related to P. graminis and P. fildesensis, respectively. DISCUSSION The Pseudomonas species exhibited the most prominent activity in vivo. Pseudomonas genus is rich in species with the potential for biocontrol of wide spectra of pathogens. Their activity is based on the production of variety of antimicrobial compounds (Dimkic et al., 2022). Also, silencing quorum sensing or quorum quenching is one of their biocontrol strategies by attenuating the virulence of the pathogen (Zhang et al., 2021). Metabarcoding analysis showed differences between treatments, mainly on the level of less presented genera. Best candidates for biocontrol of crown gall, Pseudomonas spp. R-6.10 and R-11.20 originating from the crown gall tumor, confirms the previously established hypothesis that plants are the best sources of biocontrol agents (Janisiewicz et al., 2013). CONCLUSIONS The selected Pseudomonas strains could be further tested as an alternative strategy for the biocontrol of crown gall disease and the potential involvement of the quorum quenching mechanism will be determined. Crown gall tumors have shown to be a great source of antagonistic isolates Pseudomonas sp. R-6.10 and Pseudomonas sp. R-11.20 identified according to WGS as the two new species that further needs to be described

Supplementary data for article: Dimkic, I.; Stankovic, S.; Nišavic, M.; Petkovic, M.; Ristivojevic, P.; Fira, D.; Beric, T. The Profile and Antimicrobial Activity of Bacillus Lipopeptide Extracts of Five Potential Biocontrol Strains. Frontiers in Microbiology 2017, 8 (MAY). https://doi.org/10.3389/fmicb.2017.00925

Supplementary material for: [https://doi.org/10.3389/fmicb.2017.00925]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2463

Supplementary data for the article: Dimkić, I.; Ristivojević, P.; Janakiev, T.; Berić, T.; Trifković, J.; Milojković-Opsenica, D.; Stanković, S. Phenolic Profiles and Antimicrobial Activity of Various Plant Resins as Potential Botanical Sources of Serbian Propolis. Industrial Crops and Products 2016, 94, 856–871. https://doi.org/10.1016/j.indcrop.2016.09.065

Supplementary material for: [https://doi.org/10.1016/j.indcrop.2016.09.065]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2345

Pseudomonas spp. vs. tumorigenic Rhizobium sp. – biocontrol of crown gall disease

BACKGROUND Atypical Rhizobium sp. were identified as a causative agent of crown gall on rhododendron and blueberry in Germany. Considering the lack of efficient measures against crown gall disease, evaluation of innovative biocontrol measures would greatly contribute to sustainable agriculture. OBJECTIVES Identification of candidate bacterial strains that could be employed for biological control of the novel crown gall bacteria belonging to the ″tumorigenes″ clade. METHODS Antimicrobial activity of six antagonistic Pseudomonas and Bacillus strains were tested in vitro and in vivo against pathogenic strain Rhizobium sp. rho-6.2. The in vivo experiment, using co-inoculation and preventive inoculation strategies, was performed in controlled greenhouse conditions on tomato plants as a model system in four replicas and randomized. RESULTS The whole cultures of two antagonistic Pseudomonas strains were the most efficient against pathogenic Rhizobium sp. rho-6.2 in co-inoculation strategy with reducing tumor size of 92.86%. Contrary, in the preventive treatment same Pseudomonas strains were less efficient (15.38 and 30.77%). Despite the high in vitro antimicrobial activity of Bacillus strains their in vivo activity was less pronounced in preventive treatment (up to 15.38%), while in co-inoculation strategy was more prominent (42.86%). Consortium based on Bacillus and Pseudomonas strains, was increased biocontrol activity up to 38.6% of tumor’s reduction. The selected Pseudomonas strains could be further tested as an alternative strategy for the biocontrol of crown gall disease and potential involvement of quorum quenching mechanism will be determined

Supplementary data for the article: Ristivojević, P.; Dimkić, I.; Trifković, J.; Berić, T.; Vovk, I.; Milojkovič-Opsenica, D.; Stanković, S. Antimicrobial Activity of Serbian Propolis Evaluated by Means of MIC, HPTLC, Bioautography and Chemometrics. PLoS ONE 2016, 11 (6). https://doi.org/10.1371/journal.pone.0157097

Supplementary material for: [https://doi.org/10.1371/journal.pone.0157097]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2261

INFLUENCE OF MICROBIAL AND ORGANIC FERTILIZERS ON BACTERIAL COMMUNITIES COMPOSITION DURING KEY GROWTH PHENOPHASES OF MAIZE

INTRODUCTION Maize is among the three world’s most important and widely grown cereals (Seyi-Amole & Onilude, 2021). The excessive and long-term application of agrochemicals for providing maize with essential nutrients, required for the development of all growth phenophases and for yield enhancement, leaves adverse consequences for human health and the environment (Khaliq et al., 2004). Although their use is inevitable to meet the increasing demand of the growing human population for a healthy food supply, organic fertilizers and biofertilizers (microbial fertilizers) are becoming recognized as effective, economically feasible, and environmentally sound alternatives for sustainable agriculture (Lawal & Babalola, 2014; Hui et al., 2017; Mahanty et al., 2017). The main objective of this study was to evaluate the influence of the microbial inoculant Phytobiotic (PHY), containing a consortium of Bacillus subtilis sp. subtilis and Microbacterium sp., on native maize microbiome during key growth phenophases (seedling, flowering, and harvesting) under field conditions, as well as to compare whether differences in efficacy between PHY, poultry manure (PM) and their combination (PHY_PM) exist, based on yield parameters. STATE OF THE ART Seeds, roots, and soil samples were taken for metabarcoding analysis during four growth phenophases (I-IV). Samples of uninoculated seeds and soil, poultry manure, and seeds inoculated with PHY were primarily taken before sowing (phenophase I). Further, during the growing season [phenophases II (seedling), III (flowering), and IV (harvesting)] the effect of PHY, PM, and PHY_PM on maize seeds, roots, and soil microbiome was evaluated in relation to concurrently sampled negative controls. A total DNA from the collected samples was isolated, amplified with primers 515F/ 806R targeting the V4 region of the 16S rRNA, and subjected to next-generation sequencing (NGS). The obtained sequence data were bioinformatically processed and used for the evaluation of alpha and beta diversity. Yield and associated parameters (number of grown and fallen/broken plants, rating fence, plant vigor, the occurrence of Ustilago sp., and grain moisture) were evaluated after harvest. RESULTS Seeds exibited lower bacterial diversity compared to the soil, root, and manure samples.The most abundant taxon in uninoculated seeds pre-harvest was Pantoea, while in seeds treated with PHY the most abundant was Acinetobacter, followed by Pantoea, Pseudomonas, and Bacillus. After harvest, Pantoea and Pseudomonas prevearled in seeds. Soil bacterial communities mostly remained unchanged, regardless of the treatment (PHY, PM, and PHY_PM) applied or the tested phenophase, with uncultured Gaiellales and Bacillus being the most abundant. Contrarily, root bacterial communities differed in distribution and relative abundance of different taxa between phenophases and between treatments. The most abundant taxa in roots during the inial phenophase (II) was Pseudomonas. In the flowering phenophase (III), Bacillus prevealed with two to three times higher relative abundance in treatments with PHY or PM compared to the negative control, while Lechevalieria dominated in harvesting phenophase (IV). A statistically significant increase in maize yield was obtained in the treatment with PHY, with an average value of 650 kg/ha compared to the negative control. The lowest yield was obtained in the treatment with PM. DISCUSSION The prevalence of Acinetobacter, Pantoea, Pseudomonas, and Bacillus in seeds treated with PHY pre-harvest, indicates that treatment with PHY is highly beneficial considering the known plant growth promoting potential of these genera, that were also previously confirmed as core maize inhabitants (Mehta et al., 2021). As core members, Pantoea and Pseudomonas remained present after harvest. The benefit of the application of Bacillus-based fertilizers to soil is the enhancement of the plant-available forms of nutrients and the inducement of pest and pathogens defense systems (Radhakrishnan, et al., 2017). It is of crucial importance that none of the three treatments applied in this study affected the composition of the indigenous soil bacterial communities during four tested phenophases, which is highly important when selecting suitable agricultural practices. Shifts in root microbiome over maize growth could be related to the production of different root metabolites over the growing season (Bourceret et al., 2022). Roots were especially rich with genera (Pseudomonas, Stenotrophomonas, Sphingobacterium, Achromobacter) known as phosphate solubilizers (Mehta et al., 2021). Furthermore, Bacillus was dominant in roots in flowering phenophase. This genus is known for its wide spectrum of beneficial effects on plants, like phosphate solubilization, biosynthesis of growth hormones, antimicrobial activity, induction of systemic resistance, etc. (Dimkic et al., 2022). CONCLUSIONS Considering the above-mentioned effect of PHY on maize yield incensement, its non-disruption effect on the core microbiome, and the positive effect on enhancing the presence of beneficial bacterial genera, this microbial inoculant could be proposed as a promising alternative to chemicals and organic fertilizers in maize cultivation

Fenotipska i genetička obilježja osjetljivih i višestruko otpornih izolata Pseudomonas aeruginosa u južnoj Srbiji

Drug resistance of Pseudomonas aeruginosa is a leading problem in hospital infections. The aim of this study was to determine the best molecular genetic discrimination method for Pseudomonas spp. isolates among 94 outpatients and inpatients and see their grouping by phenotype characteristics (biofilm formation, frequency of serotypes, pigmentation, production of different class of beta-lactamases, and susceptibility to different antibiotic classes) and genotype. The most common serotypes were P1, P6, and P11, while co-productions of pyoverdine and pyocyanin were observed in 70 % of isolates. A total of 77.66 % isolates were mostly weak and moderate biofilm producers. Isolates were susceptible to colistin (100 %), aztreonam (97.87 %), imipenem (91.49 %), doripenem (90.43 %), and meropenem (84.04 %). MICs values confirmed susceptibility to ceftazidime and cefepime and singled out meripenem as the most effective inhibitor. Most isolates were resistant to aminoglycosides and fluoroquinolones. Only two isolates produced ESBL, eight were carbapenemase producers, and five isolates produced MBLs. Twenty-nine isolates were multidrug-resistant; 82.8 % of which produced both pigments, 58.3 % were non-typeable, while the P6 and P11 serotypes were equally distributed (16.7 %). Thirteen MDR isolates were strong enzyme producers. RAPD PCR analysis using primer 272 proved the best at discriminatory fingerprinting for Pseudomonas isolates, as it allocated 12 clusters. A correlation between DNA patterns and antibiotic resistance, production of pigments, serotypes distribution, and biofilm formation was not observed, and only confirmed higher genetic heterogeneity among P. aeruginosa isolates, which suggests that other molecular methods are needed to reveal potential relations between genotypic patterns and phenotypic characteristics.Antibiotska rezistencija Pseudomonas aeruginosa vodeći je problem u bolničkim infekcijama. Cilj ovoga istraživanja bio je utvrditi najbolju diskriminatorno molekularno-genetičku metodu među 94 ambulantna i bolnička Pseudomonas spp. izolata kako bi se uvidjelo njihovo grupiranje u smislu različitih fenotipskih obilježja (stvaranje biofilma, učestalost serotipova, pigmentacija, proizvodnja različitih klasa beta-laktamaza i osjetljivost na različite skupine antibiotika) u skladu s genotipom. Najčešći serotipovi bili su P1, P6 i P11, a proizvodnja i pioverdina i piocijanina primijećena je kod 70 % izolata. Ukupno 77,66 % izolata uglavnom je iskazalo slabu i umjerenu proizvodnju biofilma. Izolati su bili osjetljivi na kolistin (100 %), aztreonam (97,87 %), imipenem (91,49 %), doripenem (90,43 %) i meropenem (84,04 %). Vrijednosti MIC-ova potvrdile su podložnost izolata ceftazidimu i cefepimu, a izdvojile su meropenem kao najučinkovitiji inhibitor. Većina izolata bila je otporna na aminoglikozid i fluorokinolon. Samo dva izolata proizvela su ESBL, osam izolata sintetiziralo je karbapenemaze, a pet izolata imalo je sposobnost proizvodnje MBL-a. Dvadeset devet izolata bilo je višestruko rezistentno na antibiotike, od kojih je 82,8 % proizvodilo oba pigmenta, 58,3 % bili su netipabilni, a serotipovi P6 i P11 bili su podjednako zastupljeni među njima (16,7 %). Trinaest MDR izolata bili su snažni proizvođači enzima. RAPD PCR analiza korištenjem 272 početnica pokazala se kao najbolja diskriminatorna metoda otiskom prsta (fingerprinting) za Pseudomonas izolate, izdvajajući čak 12 različitih klastera. U ovom istraživanju nije zabilježena povezanost između DNA obrazaca i otpornosti na antibiotike, proizvodnje pigmenata, distribucije serotipova i stvaranja biofilma, što potvrđuje puno veću genetičku heterogenost unutar samih izolata P. aeruginosa, pod čim se podrazumijeva uključivanje drugih molekularnih metoda u otkrivanju potencijalnih odnosa između genetičkih obrazaca i fenotipskih obilježja