Purpose
Flotation tailings represent an extremely unfriendly substrate for plant colonization due to toxic metal concentrations and marked macronutrient deficiencies. The perennial grass
Miscanthus
x
...giganteus
J.M.Greef & Deuter ex Hodk. & Renvoize was successfully cultivated in this infertile substrate for two years. Our aim was to identify composition of its rhizosphere bacterial community and to analyze the effects of the selected rhizobacteria on plant growth, root development, metal and P uptake.
Methods
Using the cultivation-dependent method, 75 isolates were collected from the rhizosphere and six rhizobacterial strains were selected for further characterization based on morphological and biochemical differences. The plant rhizomes were inoculated with the consortium of rhizobacteria and cultivated in the flotation tailings substrate.
Results
Detected bacterial strains were characterized as metal-resistant and plant growth-promoting rhizobacteria (PGPR) because of their metal tolerance (NiCl
2
, Pb(C
2
H
3
O
2
)
2
, CuSO
4
, NaAsO
2
, MnCl
2
) and some or all of the plant growth-promoting (PGP) properties (indole-3-acetic acid and siderophore production, 1-aminocyclopropane-1-carboxylic acid deaminase activity and phosphate solubilization). PGPR mitigated the negative effects of high metal concentrations and macronutrient deficiency as shown by stimulated lateral roots development, increased root hair length, plant below and above ground biomass yield, higher plant P uptake and metal accumulation rate.
Conclusions
The isolated PGPR strains could be used in PGP-bacteria assisted phytoremediation of flotation tailings and metal polluted soils by
M.
x
giganteus
. Their PGP effects on various metal-tolerant target plant species in the respective substrate remain to be verified.
Fire blight disease, caused by plant pathogenic bacteria
Erwinia amylovora
represents a global threat for pome fruit industry, affecting yield and causing serious economic damage. During six year ...period (2011–2016), a total of 45 isolates typical for
E. amylovora
were isolated from four different host plants (apple, pear, quince and medlar) in 21 localities within four Serbian regions (Southeast, Central, West, and North). Isolates were primarily identified using biochemical and serological tests, polymerase chain reaction (PCR) with species-specific primer pair (A/B) and their pathogenicity. Polyclonal antibodies used in DAS ELISA and IF were found to be specific and sensitive enough for rapid
E. amylovora
detection. In virulence test performed on immature pear fruitlets, higher variability (CV) was found between regions than among hosts from which isolates originated. Slighter virulence of isolates originated from North region of Serbia (Vojvodina) was found in comparison to the isolates from Central Serbia, which were the most variable. Principal components analysis (PCA) segregated quince as a host among the most prevalent isolates in group of highly virulent, but failed to identify difference related to geographic origin of Serbian isolates. The same results enabled multivariate statistical analysis. Different DNA fingerprinting techniques such as repetitive element sequence based PCR (rep-) with BOX, ERIC, REP, GTG
5
and SERE primers, then Random Amplified Polymorphic DNA (RAPD) with M13-PCR primer revealed genetic homogeneity among all Serbian
E. amylovora
isolates. ERIC, SERE and M13 primers generated the most complex and visually perceptible patters. Restriction fragment length polymorphism analysis (RFLP) of
gyrA
and
recA
genes, digested with three restriction endonucleases
Bst
UI,
BgI
II and
Bsm
I and
rpoS
gene digested with
Bst
UI gave the unuque, characteristic patterns for all Serbian isolates.
Plant pathogenic bacteria from the genera
Pectobacterium
and
Dickeya
in association with potato cause blackleg and tuber soft rot disease. A metabarcoding was used to analyze the microbial ...communities of soft-rotted tubers and their geocaulospheres. In September 2018, samples collected from the blackleg-infected field (58 ha) in the Bačka region (Serbia) reached 45% disease incidence. Next-generation sequencing Illumina MiSeq platform was used to obtain 16S rRNA sequences from six tested tubers (with and without soft rot symptom) and the surrounding geocaulosphere. Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria were present in all samples, with Proteobacteria being the most prevalent, especially in tubers without soft rot symptoms. In all tested samples, species of the
Pectobacterium
(
P. aroidearum
,
P. atrosepticum
,
P. carotovorum
, and
P. polaris
) were detected but were more represented in tubers without symptoms. However, the genus
Dickeya
was at the limit of detection or not detected at all.
Acinetobacter
was the most dominant in tubers with soft rot, while
Pseudomonas
and
Enterobacter
were the most abundant in tubers without symptoms. The genera
Bacteroides
and
Dysgonomonas
with starch-degradable features were almost exclusively present in soft rotted tubers and their corresponding geocaulospheres. The most represented genera in the geocaulosphere sample associated with no-symptom tubers were
Gaiella
,
Sphingomonas
,
Sphingobium
,
Gemmatimonas
, and
Geminicoccus
, which include species with confirmed biocontrol potential
.
This study indicates that the soft rot maceration process arises due to complex interactions between plant pathogens and other endophytic bacteria.
Abstract Aims This study aimed to evaluate the potential of endophytic plant growth-promoting bacteria (PGPB), Pseudomonas putida A32, to mitigate drought stress in two bell pepper genotypes, Amfora ...19 and Amfora 26, and to assess the genotype-specific responses to bacterial treatment. Methods and results The isolate P. putida A32 was selected for its remarkable beneficial properties, exhibiting 13 out of 14 traits tested. Under drought conditions, Amfora 26 showed increased relative water content (RWC) and decreased H2O2 and malondialdehyde following bacterial treatment, while Amfora 19 exhibited enhanced growth parameters but responded less to bacterial treatment regarding drought parameters. However, Amfora 19 displayed inherent drought tolerance mechanisms, as indicated by lower stress parameters compared to Amfora 26. Conclusions The study emphasises the importance of genotype-specific responses to PGPB treatment and the mechanisms of drought tolerance in peppers. P. putida A32 effectively mitigated drought stress in both genotypes, with differential responses influenced by plant genotype. Our study confirmed our initial hypothesis that Amfora 19, as a genotype tolerant to biotic stress, is also more tolerant to abiotic stress. Understanding these interactions is crucial for the development of customised strategies to improve plant productivity and tolerance to drought.
•Bacillus group are well known as producers of a wide array of antimicrobials.•Most important molecules are non-ribosomally synthesized peptides and lipopeptides.•Very strong antimicrobial activity ...was confirmed through numerous studies.•Bacillus lipopeptides have strong impact through direct antibiosis on plant pathogens.
Bacteria from the Bacillus group are microorganisms that inhabit a large number of different habitats. They are well known as producers of a wide array of antagonistic compounds of different structures, having between 5 to 8% of the total genome devoted to biosynthesis of secondary metabolites. Most important bioactive molecules from the genus Bacillus are non-ribosomally synthesized peptides and lipopeptides, polyketide compounds, bacteriocins and siderophores. Lipopeptides from Bacillus have very complex mechanisms of biosynthesis catalyzed by non-ribosomal peptide synthetases (NRPSs), large enzyme complexes with modular structure, with each module being in charge for the incorporation of a particular amino acid. In general, they have a broad spectrum of antagonistic activity against plant pathogenic bacteria, fungi and viruses. Most important molecules from this group, circular lipopeptides from surfactin, iturin and fengycin families affect the target cells on the membrane level. Bacillus strains exhibit their biocontrol capacity predominantly through inhibitory activity on the growth of plant pathogens, as well as inducing systemic resistance in plants and competing for ecological niches with plant pathogens. Our previous studies showed the presence of multiple biosynthetic operons for synthesis of non-ribosomal lipopeptides in the collection of natural isolates of Bacillus, with many strains having more than one of them. Several strains of Bacillus sp. that we have recently characterized showed very strong antibacterial and antifungal activity against phytopathogens. The PCR analysis showed the presence of biosynthetic operons for iturin, bacillomycin, fengycin and surfactin in tested strains. Measurement of the kinetics of production of antimicrobial substances showed that, in most cases, synthesis started at the beginning of exponential phase of growth, reaching the maximum of antimicrobial activity at the beginning of the stationary growth phase and stayed at this level for the whole duration of observed period. Preparations of cell-free supernatants of tested strains were active against many fungal and bacterial pathogens, in vitro and in vivo. Mass spectrometry and HPTLC bioautography analysis of purified compounds confirmed the presence of lipopeptides of mentioned families, hence confirming the biocontrol capacity of Bacillus isolates.
In this study the efficacy of two different methods for extracting lipopeptides produced by five
strains-ethyl acetate extraction, and acid precipitation followed by methanol extraction-was ...investigated using mass spectrometry. High performance thin layer chromatography (HPTLC) was also used for the simultaneous separation of complex mixtures of lipopeptide extracts and for the determination of antimicrobial activity of their components. The mass spectra clearly showed well-resolved groups of peaks corresponding to different lipopeptide families (kurstakins, iturins, surfactins, and fengycins). The ethyl acetate extracts produced the most favorable results. The extracts of SS-12.6, SS-13.1, and SS-38.4 showed the highest inhibition zones. An iturin analog is responsible for the inhibition of
and
phytopathogenic strains. HPTLC bioautography effectively identified the active compounds from a mixture of lipopeptide extracts, proving
its potential for use in direct detection and determination of antimicrobials. In the test of potential synergism among individual extracts used in different mixtures, stronger antimicrobial effects were not observed. Biochemical and phylogenetic analysis clustered isolates SS-12.6, SS-13.1, SS-27.2, and SS-38.4 together with
, while SS-10.7 was more closely related to
.
The present study provides a new insight into the existing Xanthomonas arboricola pv. pruni (Xap) bacterial population originating from peach and apricot in two Western Balkans countries (Serbia and ...Montenegro). Multilocus sequence typing and analysis of the sequences of nine housekeeping genes revealed homology between the tested Xap strains as well as with the European population of this bacterium. The tested strains share the same haplotype (Haplotype I) with the Xap strains from Italy, France, Spain, United States, Australia and Brazil. The revealed single nucleotide change (G ↔ C) in the sequences of the gyrB1 gene differentiates Haplotype I from Haplotype II (Xap from South Korea, New Zealand, Argentina and Uruguay). The detached‐leaf bioassay results confirmed differences in virulence between strains originating from peach and apricot towards Prunus armeniaca (apricot), indicating host specialization of the apricot strain towards this host. For the first time, immunity of P. fruticosa (European ground cherry) to Xap was established. According to the AUDPC, PCA and cluster analysis, other Prunus spp. were classified as having low susceptibility (P. mahaleb, P. cerasus and P. avium), as susceptible (P. domestica) and as highly susceptible (P. persica, P. dulcis, P. cerasifera and P. spinosa). Xap strains were also found to be susceptible to 10 tested antibiotics. This study provides valuable knowledge on the Xap population from stone fruit grown in the Western Balkans region as well as the source of immunity, which could serve as a starting point for breeding Prunus cultivars and could be used as the main control strategy.
Peach and apricot Xanthomonas arboricola pv. pruni (Serbia and Montenegro) compose a genetically homogeneous group with European population; host specialization of the apricot strain and Prunus fruticosa immunity were determined.
New information has come to light about the biological activity of propolis and the quality of natural products which requires a rapid and reliable assessment method such as High Performance ...Thin-Layer Chromatography (HPTLC) fingerprinting. This study investigates chromatographic and chemometric approaches for determining the antimicrobial activity of propolis of Serbian origin against various bacterial species. A linear multivariate calibration technique, using Partial Least Squares, was used to extract the relevant information from the chromatographic fingerprints, i.e. to indicate peaks which represent phenolic compounds that are potentially responsible for the antimicrobial capacity of the samples. In addition, direct bioautography was performed to localize the antibacterial activity on chromatograms. The biological activity of the propolis samples against various bacterial species was determined by a minimum inhibitory concentration assay, confirming their affiliation with the European poplar type of propolis and revealing the existence of two types (blue and orange) according to botanical origin. The strongest antibacterial activity was exhibited by sample 26 against Staphylococcus aureus, with a MIC value of 0.5 mg/mL, and Listeria monocytogenes, with a MIC as low as 0.1 mg/mL, which was also the lowest effective concentration observed in our study. Generally, the orange type of propolis shows higher antimicrobial activity compared to the blue type. PLS modelling was performed on the HPTLC data set and the resulting models might qualitatively indicate compounds that play an important role in the activity exhibited by the propolis samples. The most relevant peaks influencing the antimicrobial activity of propolis against all bacterial strains were phenolic compounds at RF values of 0.37, 0.40, 0.45, 0.51, 0.60 and 0.70. The knowledge gained through this study could be important for attributing the antimicrobial activity of propolis to specific chemical compounds, as well as the verification of HPTLC fingerprinting as a reliable method for the identification of compounds that are potentially responsible for antimicrobial activity. This is the first report on the activity of Serbian propolis as determined by several combined methods, including the modelling of antimicrobial activity by HPTLC fingerprinting.
Brown rot caused by Monilinia spp. is one of the main causes of pre- and postharvest losses in stone and pome fruit production. The use of beneficial microorganisms is considered one of the most ...promising, safe and effective alternative methods for controlling these pathogens. This study aimed to investigate the antagonistic potential of 33 Bacillus spp. strains, in order to identify the best candidate for brown rot biocontrol. Strains identified as Bacillus amyloliquefaciens B-241 and Bacillus subtilis B-313 and B-358 were chosen for further ex situ studies on detached apple fruit. The efficacy of B-241 (87.1–93.7%) did not differ significantly from a commercially available synthetic fungicide (p > 0.05). The putative mode of action of B. amyloliquefaciens B-241 against Monilinia species is competition for nutrients and antibiosis. The ethyl acetate extract of the strain, applied at 5 and 12.5 mg/mL, was bioactive in vitro and ex situ. A HPLC analysis confirmed the presence of surfactin and bacillomycin D in the extract. However, before developing a shelf-stable product and commercial production, the spectrum and efficacy on a larger scale of the B-241 strain should be determined, and its efficacy in combination with commercial biofungicides and fungicides tested in vivo.
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