Diversity of antagonistic bacteria isolated from medicinal plant Peganum harmala L.

The antimicrobial activity of plant extract of Peganum harmala, a medicinal plant has been studied already. However, knowledge about bacterial diversity associated with different parts of host plant antagonistic to different human pathogenic bacteria is limited. In this study, bacteria were isolated from root, leaf and fruit of plant. Among 188 bacterial isolates isolated from different parts of the plant only 24 were found to be active against different pathogenic bacteria i.e. Escherichia coli, Methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecium, Enterococcus faecalis and Pseudomonas aeruginosa. These active bacterial isolates were identified on the basis of 16S rRNA gene analysis. Total population of bacteria isolated from plant was high in root, following leaf and fruit. Antagonistic bacteria were also more abundant in root as compared to leaf and fruit. Two isolates (EA5 and EA18) exhibited antagonistic activity against most of the targeted pathogenic bacteria mentioned above. Some isolates showed strong inhibition for one targeted pathogenic bacterium while weak or no inhibition for others. Most of the antagonistic isolates were active against MRSA, following E. faecium, P. aeruginosa, E. coli and E. faecalis. Taken together, our results show that medicinal plants are good source of antagonistic bacteria having inhibitory effect against clinical bacterial pathogens.     

Bacterial community composition and chitinase gene diversity of vermicompost with antifungal activity

Bacterial communities and chitinase gene diversity of vermicompost (VC) were investigated to clarify the influence of earthworms on the inhibition of plant pathogenic fungi in VC. The spore germination of Fusarium moniliforme was reduced in VC aqueous extracts prepared from paper sludge and dairy sludge (fresh sludge, FS). The bacterial communities were examined by culture-dependent and -independent analyses. Unique clones selected from 16S rRNA libraries of FS and VC on the basis of restriction fragment length polymorphism (RFLP) fell into the major lineages of the domain bacteria Proteobacteria, Bacteroidetes, Verrucomicrobia, Actinobacteria and Firmicutes. Among culture isolates, Actinobacteria dominated in VC, while almost equal numbers of Actinobacteria and Proteobacteria were present in FS. Analysis of chitinolytic isolates and chitinase gene diversity revealed that chitinolytic bacterial communities were enriched in VC. Populations of bacteria that inhibited plant fungal pathogens were higher in VC than in FS and particularly chitinolytic isolates were most active against the target fungi.     

Isolation and characterization of plant growth promoting endophytic diazotrophic bacteria from Korean rice cultivars

We have isolated 576 endophytic bacteria from the leaves, stems, and roots of 10 rice cultivars and identified 12 of them as diazotrophic bacteria using a specific primer set of nif gene. Through 16S rDNA sequence analysis, nifH genes were confirmed in the two species of Penibacillus, three species of Microbacterium, three Bacillus species, and four species of Klebsiella. Rice seeds treated with these plant growth-promoting bacteria (PGPB) showed improved plant growth, increased height and dry weight and antagonistic effects against fungal pathogens. In addition, auxin and siderophore producing ability, and phosphate solubilizing activity were studied for the possible mechanisms of plant growth promotion. Among 12 isolates tested, 10 strains have shown higher auxin producing activity, 6 isolates were confirmed as strains with high siderophore producing activity while 4 isolates turned out to have high phosphate-solubilizing activity. These results strongly suggest that the endophytic diazotrophic bacteria characterized in this study could be successfully used to promote plant growth and inducing fungal resistance in plants.     

The N-Glycan Cluster from Xanthomonas campestris pv. campestris: A TOOLBOX FOR SEQUENTIAL PLANT N-GLYCAN PROCESSING*

N-Glycans are widely distributed in living organisms but represent only a small fraction of the carbohydrates found in plants. This probably explains why they have not previously been considered as substrates exploited by phytopathogenic bacteria during plant infection. Xanthomonas campestris pv. campestris, the causal agent of black rot disease of Brassica plants, possesses a specific system for GlcNAc utilization expressed during host plant infection. This system encompasses a cluster of eight genes (nixE to nixL) encoding glycoside hydrolases (GHs). In this paper, we have characterized the enzymatic activities of these GHs and demonstrated their involvement in sequential degradation of a plant N-glycan using a N-glycopeptide containing two GlcNAcs, three mannoses, one fucose, and one xylose (N₂M₃FX) as a substrate. The removal of the α-1,3-mannose by the α-mannosidase NixK (GH92) is a prerequisite for the subsequent action of the β-xylosidase NixI (GH3), which is involved in the cleavage of the β-1,2-xylose, followed by the α-mannosidase NixJ (GH125), which removes the α-1,6-mannose. These data, combined to the subcellular localization of the enzymes, allowed us to propose a model of N-glycopeptide processing by X. campestris pv. campestris. This study constitutes the first evidence suggesting N-glycan degradation by a plant pathogen, a feature shared with human pathogenic bacteria. Plant N-glycans should therefore be included in the repertoire of molecules putatively metabolized by phytopathogenic bacteria during their life cycle.     

Biodiversity of endophytic fungi in different leaf ages of Calotropis procera and their antimicrobial activity

Calotropis procera has many important medicinal properties with proven pharmacological potential. Some of these properties may be mediated by its fungal endophytes. This study analyzed, for the first time, the community of endophytic fungi of C. procera outside its region of origin. A total of 156 fungal isolates distributed across 19 taxa were obtained from 468 fragments of C. procera leaves at different stages of maturation. The rate of endophyte colonization increased with the leaf age/development. The dominant species of endophytic fungi of C. procera introduced in Northeast Brazil were different from those found in studies on the same species and other species of the same genus in native regions. The dominant endophyte was Phaeoramularia calotropidis (63.5 %), followed by Guignardia bidwellii (21.1 %). Six isolates of endophytic fungi showed antimicrobial activity against human pathogenic micro-organisms and one plant pathogenic fungus. The antibacterial activity was more intense than the antifungal activity. The endophytic Curvularia pallescens (URM 6048) stood out inhibited Gram-positive bacteria, Staphylococcus aureus, Streptococcus pyogenes, the plant pathogenic fungus Colletotrichum dematium. Ecological and biotechnological aspects of endophytic mycota are discussed.     

Structural and phylogenetic analyses of the GP42 transglutaminase from Phytophthora sojae reveal an evolutionary relationship between oomycetes and marine Vibrio bacteria

Transglutaminases (TGases) are ubiquitous enzymes that catalyze selective cross-linking between protein-bound glutamine and lysine residues; the resulting isopeptide bond confers high resistance to proteolysis. Phytophthora sojae, a pathogen of soybean, secretes a Ca²⁺-dependent TGase (GP42) that is activating defense responses in both host and non-host plants. A GP42 fragment of 13 amino acids, termed Pep-13, was shown to be absolutely indispensable for both TGase and elicitor activity. GP42 does not share significant primary sequence similarity with known TGases from mammals or bacteria. This suggests that GP42 has evolved novel structural and catalytic features to support enzymatic activity. We have solved the crystal structure of the catalytically inactive point mutant GP42 (C290S) at 2.95 Å resolution and identified residues involved in catalysis by mutational analysis. The protein comprises three domains that assemble into an elongated structure. Although GP42 has no structural homolog, its core region displays significant similarity to the catalytic core of the Mac-1 cysteine protease from Group A Streptococcus, a member of the papain-like superfamily of cysteine proteases. Proteins that are taxonomically related to GP42 are only present in plant pathogenic oomycetes belonging to the order of the Peronosporales (e.g. Phytophthora, Hyaloperonospora, and Pythium spp.) and in marine Vibrio bacteria. This suggests that a lateral gene transfer event may have occurred between bacteria and oomycetes. Our results offer a basis to design and use highly specific inhibitors of the GP42-like TGase family that may impair the growth of important oomycete and bacterial pathogens.     

NtPR1a regulates resistance to Ralstonia solanacearum in Nicotiana tabacum via activating the defense-related genes

Pathogenesis-related proteins (PRs) are associated with the development of systemic acquired resistance (SAR) against further infection enforced by fungi, bacteria and viruses. PR1a is the first PR-1 member that could be purified and characterized. Previous studies have reported its role in plants’ resistance system against oomycete pathogens. However, the role of PR1a in Solanaceae plants against the bacterial wilt pathogen Ralstonia solanacearum remains unclear. To assess roles of NtPR1a in tobacco responding to R. solanacearum, we performed overexpression experiments in Yunyan 87 plants (a susceptible tobacco cultivar). The results illuminated that overexpression of NtPR1a contributed to improving resistance to R. solanacearum in tobacco Yunyan 87. Specifically speaking, NtPR1a gene could be induced by exogenous hormones like salicylic acid (SA) and pathogenic bacteria R. Solanacearum. Moreover, NtPR1a-overexpressing tobacco significantly reduced multiple of R. solanacearum and inhibited the development of disease symptoms compared with wild-type plants. Importantly, overexpression of NtPR1a activated a series of defense-related genes expression, including the hypersensitive response (HR)-associated genes NtHSR201 and NtHIN1, SA-, JA- and ET-associated genes NtPR2, NtCHN50, NtPR1b, NtEFE26, and Ntacc oxidase, and detoxification-associated gene NtGST1. In summary, our results suggested that NtPR1a-enhanced tobacco resistance to R. solanacearum may be mainly dependent on activation of the defense-related genes.     

An endophytic bacterium isolated from Panax ginseng C.A. Meyer enhances growth,reduces morbidity,and stimulates ginsenoside biosynthesis

Ginseng (Panax ginseng C.A. Meyer) is known for its therapeutically useful ginsenosides that have anticancer and other pharmacological effects. However, its low levels in plants and the high costs of chemical synthesis make ginsenosides commercially non-viable; as such, strategies for increasing ginsenoside yield are of great interest. The present study reports the isolation of eight novel endophytic bacteria from ginseng leaves, the highest ginsenoside concentration of microbial transformed strain was identified as Paenibacillus polymyxa. Inoculation of ginseng plants with P. polymyxa by foliar application combined with irrigation enhanced plant growth parameters, reduced morbidity, and increased plant concentration of the ginsenosides (Rg₁, Re, Rf, Rb₁, Rg₂, Rb₂, Rb₃, and Rd) in field experiments. These results indicate that P. polymyxa isolated from ginseng is a beneficial endophytic bacterium with biocontrol properties that can enhance the yield and quality of this medicinal plant.     

The Potential Anti-Herbivory Role of Microorganisms on Plant Thorns

Thorns, spines and prickles are some of the anti-herbivore defenses that plants have evolved. They were recently found to be commonly aposematic (warning coloration). However, the physical anti-herbivore defense executed by these sharp structures seems to be only the tip of the iceberg. We show that thorns of various plant species commonly harbor an array of aerobic and anaerobic pathogenic bacteria including Clostridium perfringens the causative agent of the life-threatening gas gangrene, Bacillus anthracis, and Pantoea agglomerans. Septic inflammation caused by plant thorn injury can result not only from bacteria. Medical literature indicates that thorns, spines or prickles also introduce pathogenic fungi into animals or humans. Dermatophytes that cause subcutaneous mycoses are unable to penetrate the skin and must be introduced into the subcutaneous tissue by a puncture wound. The common microorganism-thorn combinations seem to have been an important contributor to the fact that so many plant thorns are aposematically colored, as a case of convergent evolution of aposematism in these organisms.Key Words: aposematism, herbivory, pathogen, spine, thorn, bacillus anthracis, clostridium perfringens, sporotrichosis, Mycetoma, subcutaneous mycotic disease     

Saprozoic Nematodes as Carriers and Disseminators of Plant Pathogenic Bacteria

The plant pathogenic bacteria Agrobacterium tumefaciens (Smith and Townsend) Conn. (strain 5-14 Deep), Erwinia amylovora (Burill) Winslow et al., E. carotovora (Jones) Holland and Pseudornonas phaseolicola (Burk.) Dows. (ICPB-PM3) and the red-pigmented non-pathogen Serratia marcescens Bizio were hosts for the saprozoic nematode Pristionchus Iheritieri (Maupas, 1919) Paramonov. Viable bacteria survived passage through the nematode and produced typical colonies on nutrient agar plates. Female nematodes ingested more bacterial cells and retained them longer than did males. It was hypothesized saprozoic nematodes may disseminate pathogenic bacteria to new infection courts.     

Evaluation of diffusion and dilution methods to determine the antibacterial activity of plant extracts

The aim of this study was to evaluate diffusion and dilution methods for determining the antibacterial activity of plant extracts and their mixtures. Several methods for measurement of the minimal inhibitory concentration (MIC) of a plant extract are available, but there is no standard procedure as there is for antibiotics. We tested different plant extracts, their mixtures and phenolic acids on selected gram-positive (Staphylococcus aureus, Bacillus cereus, and Listeria monocytogenes) and gram-negative bacteria (Escherichia coli O157:H7, Salmonella Infantis, Campylobacter jejuni, Campylobacter coli) with the disk diffusion, agar dilution, broth microdilution and macrodilution methods. The disk diffusion method was appropriate only as a preliminary screening test prior to quantitative MIC determination with dilution methods. A comparison of the results for MIC obtained by agar dilution and broth microdilution was possible only for gram-positive bacteria, and indicated the latter as the most accurate way of assessing the antimicrobial effect. The microdilution method with TTC (2,3,5-triphenyl tetrazolium chloride) or INT (2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride) to indicate the viability of aerobic bacteria was found to be the best alternative approach, while only ATP determination was appropriate for microaerophilic Campylobacter spp. Using survival curves the kinetics of bacterial inactivation on plant extract exposure was followed for 24 h and in this way the MIC values determined by the microdilution method were confirmed as the concentrations of extracts that inhibited bacterial growth. We suggest evaluation of the antibacterial activity of plant extracts using the broth microdilution method as a fast screening method for MIC determination and the macrodilution method at selected MIC values to confirm bacterial inactivation. Campylobacter spp. showed a similar sensitivity to plant extracts as the tested gram-positive bacteria, but S. Infantis and E. coli O157:H7 were more resistant.     

In Vitro Antibacterial Activity of Cysteine Protease Inhibitor from Kiwifruit (Actinidia deliciosa)

The need for replacing traditional pesticides with alternative agents for the management of agricultural pathogens is rising worldwide. In this study, a cysteine proteinase inhibitor (CPI), 11 kDa in size, was purified from green kiwifruit to homogeneity. We examined the growth inhibition of three plant pathogenic Gram-negative bacterial strains by kiwi CPI and attempted to elucidate the potential mechanism of the growth inhibition. CPI influenced the growth of phytopathogenic bacteria Agrobacterium tumefaciens (76.2 % growth inhibition using 15 μM CPI), Burkholderia cepacia (75.6 % growth inhibition) and, to a lesser extent, Erwinia carotovora (44.4 % growth inhibition) by inhibiting proteinases that are excreted by these bacteria. Identification and characterization of natural plant defense molecules is the first step toward creation of improved methods for pest control based on naturally occurring molecules.     

Use of endophytes as biocontrol agents

Plant diseases, caused by various microorganisms, including viruses, bacteria, fungi, protozoa and nematodes, affect agricultural practices and result in significant crop losses. Fungal pathogens are the major cause of plant diseases and infect most plants. Agrochemicals play a significant role in plant disease management to ensure a sustainable and productive agricultural system. However, the intensive use of chemicals has adverse effects on humans and ecosystem functioning and also reduces agricultural sustainability. A sustainable agriculture is achieved through reduction or elimination of fertilizers and agrochemicals, resulting in minimal impact to the environment. Recently, the use of antagonistic endophytes as biocontrol agents is drawing special attention as an attractive option for management of some plant diseases, resulting in minimal impact to the environment. Endophytes that resides asymptomatically within a plant, have the potential to provide a source of candidate strains for potential biocontrol applications. This review addresses biocontrol methods using endophytic fungi such as Colletotrichum, Cladosporium, Fusarium, Pestalotiopsis and Trichoderma species as an attractive option for management of some plant diseases. Potential endophytes are screened in vitro and in vivo to test their antagonistic actions by different mechanisms, including mycoparasitism, production of lytic enzymes and/or antibiotics and induction of plant defenses. Currently, efforts are being made to commercialize these biocontrol agents. A continued research pipeline consisting of screening, in vitro and in vivo testing, biomass production and commercialization of endophytes as biocontrol agents may contribute to sustainable agriculture.     

Hrp- Mutants of Pseudomonas solanacearum as Potential Biocontrol Agents of Tomato Bacterial Wilt

There have been many attempts to control bacterial wilt with antagonistic bacteria or spontaneous nonpathogenic mutants of Pseudomonas solanacearum that lack the ability to colonize the host, but they have met with limited success. Since a large gene cluster (hrp) is involved in the pathogenicity of P. solanacearum, we developed a biological control strategy using genetically engineered Hrp^- mutants of P. solanacearum. Three pathogenic strains collected in Guadeloupe (French West Indies) were rendered nonpathogenic by insertion of an ω-Km interposon within the hrp gene cluster of each strain. The resulting Hrp^- mutants were tested for their ability to control bacterial wilt in challenge inoculation experiments conducted either under growth chamber conditions or under greenhouse conditions in Guadeloupe. Compared with the colonization by a pathogenic strain which spread throughout the tomato plant, colonization by the mutants was restricted to the roots and the lower part of the stems. The mutants did not reach the fruit. Moreover, the presence of the mutants did not affect fruit production. When the plants were challenge inoculated with a pathogenic strain, the presence of Hrp^- mutants within the plants was correlated with a reduction in disease severity, although pathogenic bacteria colonized the stem tissue at a higher density than the nonpathogenic bacteria. Challenge inoculation experiments conducted under growth chamber conditions led, in some cases, to exclusion of the pathogenic strain from the aerial part of the plant, resulting in high protection rates. Furthermore, there was evidence that one of the pathogenic strains used for the challenge inoculations produced a bacteriocin that inhibited the in vitro growth of the nonpathogenic mutants.     

Antimicrobial Constituents of the Leaves of Mikania micrantha H. B. K

Background

To isolate plant-derived compounds with antimicrobial activity from the leaves of Mikania micrantha, to determine the compounds configuration, and to evaluate their antimicrobial activity against eight plant pathogenic fungi (Exserohilum turcicum, Colletotrichum lagenarium, Pseudoperonispora cubensis, Botrytis cirerea, Rhizoctonia solani, Phytophthora parasitica, Fusarium solani, and Pythium aphanidermatum,) and four plant pathogenic bacteria (gram negative bacteria: Ralstonia dolaanacearum, Xanthomonas oryzae pv. Oryzae, Xanthomonas Campestris pv. Vesicatoria, and Xanthomonas campestris pv. Citri), and four bacteria (gram positive bacteria: Staphyloccocus aureus, Bacillus subtilis, Micrococcus luteus, and Bacillus cereus).

Methods and Results

Antimicrobial constituents of the leaves of M. micrantha were isolated using bioactivity- guided fractionation. The antifungal activity of the isolated compounds was evaluated by the inhibit hypha growth method and inhibit spore germination method. Characterization of antibacterial activity was carried out using the minimum inhibitory concentrations (MICs) and the minimum bactericidal concentrations (MBCs). MIC and MBC were determined by the broth microdilution method. Six compounds – deoxymikanolide, scandenolide, dihydroscandenolide, mikanolide, dihydromikanolide, and m - methoxy benzoic acid – have been isolated from leaves of Mikania micrantha H. B. K. Deoxymikanolide, scandenolide, and dihydroscandenolide were new compounds. The result of bioassay showed that all of isolated compounds were effective against tested strains and deoxymikanolide showed the strongest activity.

Conclusions and Significance

The leaves of M. micrantha may be a promising source in the search for new antimicrobial drugs due to its efficacy and the broadest range. Meanwhile, adverse impact of M. micrantha will be eliminated.     

Comparative study of antifungal activity of two preparations of green silver nanoparticles from Portulaca oleracea extract

The green silver nanoparticles (green AgNPs) exhibit an exceptional antimicrobial property against different microbes, including bacteria and fungi. The current study aimed to compare the antifungal activities of both the crude aqueous extract of Portulaca oleracea or different preparations of green AgNPs biosynthesized by mixing that aqueous extract with silver nitrate (AgNO₃). Two preparations of the green AgNPs were synthesized either by mixing the aqueous extract of P. oleracea with silver nitrate (AgNO₃) (normal AgNPs) or either irradiation of the AgNPs, previously prepared, under ⁶⁰Co γ-ray using chitosan (gamma-irradiated AgNPs). Characterization of different AgNPs were tested by Zeta potential analyzer, Ultraviolet (UV) Visible Spectroscopy, and Fourier-Transform Infrared (FTIR) spectrometry. Three different plant pathogenic fungi were tested, Curvularia spicifera, Macrophomina phaseolina, and Bipolaris sp. The antifungal activities were evaluated by Transmission Electron Microscope (TEM) for either the crude aqueous extract of P. oleracea at three doses (25%, 50%, and 100%) or the newly biosynthesized AgNPs, normal or gamma-irradiated. With a few exceptions, the comparative analysis revealed that the irradiated green AgNPs at all three concentrations showed a relatively stronger antifungal effect than the normal AgNPs against all the three selected fungal strains. UV–visible spectroscopy of both preparations showed surface plasmon resonance at 421 nm. TEM results showed that both AgNPs were aggregated and characterized by a unique spherical shape, however, the gamma-irradiated AgNPs were smaller than the non-irradiated AgNPs (0.007–0.026 µM vs. 0.009–0.086 µM). TEM photographs of the fungal strains treated with the two AgNPs preparations showed flaccid structures, condensed hyphae, and shrunken surface compared with control cells. The data suggested that the biosynthesized P. oleracea AgNPs have antifungal properties against C. spicifera, M. phaseolina, and Bipolaris sp. These AgNPs may be considered a fungicide to protect different plants against phytopathogenic fungi.     

Comparison of Antimicrobial Activity of Essential Oils,Plant Extracts and Methylparaben in Cosmetic Emulsions: 2 Months Study

The aim of the study was to compare the preservative effectiveness of plant extracts (Matricaria chamomilla, Aloe vera, Calendula officinalis) and essential oils (Lavandulla officinalis, Melaleuca alternifolia, Cinnamomum zeylanicum) with methylparaben in cosmetic emulsions against skin microflora during 2 months of application by volunteers. Cosmetic emulsions with extracts (2.5 %), essential oils (2.5 %), methylparaben (0.4 %) or placebo were tested by 40 volunteers during 2 months of treatment. In order to determine microbial purity of the emulsions, the samples were taken after 0, 2, 4, 6 and 8 weeks of application. Throughout the trial period it was revealed that only cinnamon oil completely inhibited the growth of bacteria, yeast and mould, as compared to all other essential oils, plant extracts and methylparaben in the tested emulsions. This result shows that cinnamon oil could successfully replace the use of methylparaben in cosmetics, at the same time ensuring microbiological purity of a cosmetic product under its in-use and storage conditions.     

Associations between Vaginal Pathogenic Community and Bacterial Vaginosis in Chinese Reproductive-Age Women

Background

Bacterial vaginosis (BV) is one of the most common urogenital infections among women of reproductive age that represents shifts in microbiota from Lactobacillus spp. to diverse anaerobes. The aim of our study was to evalute the diagnostic values of Gardnerella, Atopobium, Eggerthella, Megasphaera typeI, Leptotrichia/Sneathia and Prevotella, defined as a vaginal pathogenic community for BV and their associations with vaginal pH and Nugent scores.

Methods and Findings

We investigated the vaginal pathogenic bacteria and Lactobacillus spp. with species-specific real-time quantitative PCR (qPCR) in 50 BV-positive and 50 BV-negative Chinese women of reproductive age. Relative to BV-negative subjects, a siginificant decline in Lactobacillus and an obvious increase in bacteria in the vaginal pathogenic community were observed in BV-postive subjects (P<0.05). With the exception of Megasphaera typeI, other vaginal pathogenic bacteria were highly predictable for BV with a better sensitivity and specificity. The vaginal pathogenic community was positively associated with vaginal pH and Nugent scores, while Lactobacillus spp., such as L. iners and L. crispatus was negatively associated with them (P<0.05).

Conclusions

Our data implied that the prevalance of vaginal pathogenic bacteria as well as the depletion of Lactobacillus was highly accurate for BV diagnosis. Vaginal microbiota shifts, especially the overgrowth of the vaginal pathogenic community, showed well diagnostic values in predicting BV. Postive correlations between those vaginal pathogenic bacteria and vaginal pH, Nugent score indicated the vaginal pathogenic community rather than a single vaginal microorganism, was participated in the onset of BV directly.     

Response of Endophytic Bacterial Communities in Potato Plants to Infection with Erwinia carotovora subsp. atroseptica

The term endophyte refers to interior colonization of plants by microorganisms that do not have pathogenic effects on their hosts, and various endophytes have been found to play important roles in plant vitality. In this study, cultivation-independent terminal restriction fragment length polymorphism analysis of 16S ribosomal DNA directly amplified from plant tissue DNA was used in combination with molecular characterization of isolates to examine the influence of plant stress, achieved by infection with the blackleg pathogen Erwinia carotovora subsp. atroseptica, on the endophytic population in two different potato varieties. Community analysis clearly demonstrated increased bacterial diversity in infected plants compared to that in control plants. The results also indicated that the pathogen stress had a greater impact on the bacteria population than the plant genotype had. Partial sequencing of the 16S rRNA genes of isolated endophytes revealed a broad phylogenetic spectrum of bacteria, including members of the α, β, and γ subgroups of the Proteobacteria, high- and low-G+C-content gram-positive organisms, and microbes belonging to the Flexibacter-Cytophaga-Bacteroides group. Screening of the isolates for antagonistic activity against E. carotovora subsp. atroseptica revealed that 38% of the endophytes protected tissue culture plants from blackleg disease.     

Culture-independent analysis of an endophytic core microbiome in two species of wheat: Triticum aestivum L. (cv. ‘Hondia’) and the first report of microbiota in Triticum spelta L. (cv. ‘Rokosz’)

The main goal of the study was to determine the structure of endophytic bacteria inhabiting different parts (endosperm, germ, roots, coleoptiles, and leaves) of two wheat species, Triticum aestivum L. (cv. ‘Hondia’) and Triticum spelta L. (cv. ‘Rokosz’), in order to provide new knowledge about the stability and/or changeability of the core microbiome in different plant organs. The endophytic core microbiome is associated with plants throughout their whole life cycle; however, plant organs can determine the actual endophytic community. Therefore, next generation sequencing with MiSeq Illumina technology was applied to identify the endophytic microbiome of T. aestivum and T. spelta. Bioinformatic analyses were performed with the use of the DADA2(1.8) package and R software (3.5.1).It was demonstrated that wheat, which is an important crop plant, was associated with beneficial endophytic bacteria inside the endosperms, germs, roots, leaves, and coleoptiles. Importantly, for the first time, biodiversity was recognized in the coleoptiles of the investigated wheat species. Flavobacterium, Pseudomonas and Janthinobacterium were shown to be common genera for both tested wheat cultivars. Among them, Pseudomonas was found to be the only endophytic genus accompanying both wheat species from the endosperm stage to the development of the leaf. Paenibacillus was recognized as a core genus for the ‘Hondia’ cv., whereas Pedobacter and Duganella constituted the core microbiome in the ‘Rokosz’ cv. In addition, the first insight into the unique and yet unrecognized endophytic microbiome of T. spelta is presented.