The genus '
Phytoplasma' was proposed to accommodate cell wall-less bacteria that are molecularly and biochemically incompletely characterized, and colonize plant phloem and insect vector tissues. ...This provisional classification is highly relevant due to its application in epidemiological and ecological studies, mainly aimed at keeping the severe phytoplasma plant diseases under control worldwide. Given the increasing discovery of molecular diversity within the genus '
. Phytoplasma', the proposed guidelines were revised and clarified to accommodate those '
. Phytoplasma' species strains sharing >98.65 % sequence identity of their full or nearly full 16S rRNA gene sequences, obtained with at least twofold coverage of the sequence, compared with those of the reference strain of such species. Strains sharing <98.65 % sequence identity with the reference strain but >98.65 % with other strain(s) within the same '
. Phytoplasma' species should be considered related strains to that '
. Phytoplasma' species. The guidelines herein, keep the original published reference strains. However, to improve '
. Phytoplasma' species assignment, complementary strains are suggested as an alternative to the reference strains. This will be implemented when only a partial 16S rRNA gene and/or a few other genes have been sequenced, or the strain is no longer available for further molecular characterization. Lists of '
. Phytoplasma' species and alternative reference strains described are reported. For new '
. Phytoplasma' species that will be assigned with identity ≥98.65 % of their 16S rRNA gene sequences, a threshold of 95 % genome-wide average nucleotide identity is suggested. When the whole genome sequences are unavailable, two among conserved housekeeping genes could be used. There are 49 officially published '
Phytoplasma' species, including '
. P. cocostanzaniae' and '
. P. palmae' described in this manuscript.
Plant-associated
Stenotrophomonas
isolates have great potential for plant growth promotion, especially under stress conditions, due to their ability to promote tolerance to abiotic stresses such as ...salinity or drought. The endophytic strain
Stenotrophomonas
sp. 169, isolated from a field-grown poplar, increased the growth of inoculated
in vitro
plants, with a particular effect on root development, and was able to stimulate the rooting of poplar cuttings in the greenhouse. The strain produced high amounts of the plant growth-stimulating hormone auxin under
in vitro
conditions. The comparison of the 16S rRNA gene sequences and the phylogenetic analysis of the core genomes showed a close relationship to
Stenotrophomonas chelatiphaga
and a clear separation from
Stenotrophomonas maltophilia
. Whole genome sequence analysis revealed functional genes potentially associated with attachment and plant colonization, growth promotion, and stress protection. In detail, an extensive set of genes for twitching motility, chemotaxis, flagella biosynthesis, and the ability to form biofilms, which are connected with host plant colonization, could be identified in the genome of strain 169. The production of indole-3-acetic acid and the presence of genes for auxin biosynthesis pathways and the spermidine pathway could explain the ability to promote plant growth. Furthermore, the genome contained genes encoding for features related to the production of different osmoprotective molecules and enzymes mediating the regulation of stress tolerance and the ability of bacteria to quickly adapt to changing environments. Overall, the results of physiological tests and genome analysis demonstrated the capability of endophytic strain 169 to promote plant growth. In contrast to related species, strain 169 can be considered non-pathogenic and suitable for biotechnology applications.
The invasive ascomycete
Hymenoscyphus fraxineus
has been threatening
Fraxinus excelsior
populations throughout Europe for over two decades. Since the infection and first colonization by the pathogen ...occurs in leaves, leaf-colonizing microorganisms have been discussed as a barrier and as possible biocontrol agents against the disease. To identify fungal groups with health-supporting potential, we compared the fungal microbiota of compound leaves from susceptible and tolerant ash trees in four ash stands with high
H. fraxineus
exposure. The fungal communities were analyzed both culture-independently by ITS2 amplicon sequencing and by the taxonomic classification of 1,704 isolates using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) or sequencing of the entire ITS region. The fungal community structure did not show significant differences depending on the health status. However, for several OTUs and a MALDI group, a significantly higher abundance was found in tolerant ash trees. Thus, the yeast
Papiliotrema flavescens
was significantly increased and accounted for 12.3% of the mycobiome of tolerant ashes (OTU0003), and it had also a distinctly higher abundance among the isolates. The filamentous ascomycete
Sarocladium strictum
was increased 24-fold among the isolates of tolerant trees, but its abundance was comparably low. An
in vitro
screening for the growth inhibition of the pathogen
via
cocultivation resulted in 28 yeast-like isolates and 79 filamentous fungi with antagonistic activity. A statistical cocultivation test on two
H. fraxineus
strains confirmed six of the yeast-like isolates that suppressed
H. fraxineus
significantly, from 39–50%, two of them through a fungicidal effect. The highest inhibition rates among the yeasts were found for three isolates belonging to
Aureobasidium pullulans
and
P. flavescens
. The cocultivation test of the filamentous isolates revealed higher effects compared to the yeasts. Four isolates showed significant inhibition of both
H. fraxineus
strains with a rate of 72–100%, and five further isolates inhibited only one
H. fraxineus
strain significantly. The most effective isolates were members of the genus
Cladosporium
. During the next step,
in planta
tests will be necessary to verify the efficacy of the antagonistic isolates and to assess their suitability as biocontrol agents.
'Candidatus Phytoplasma ulmi' is the agent associated with elm yellows and has been categorised in the European Union as a quarantine pathogen. For central and northern European countries, ...information on the occurrence and distribution of the pathogen and its impact on elms is scarce, so a survey of native elm trees has been conducted in Germany.
About 6500 samples from Ulmus minor, Ulmus laevis and Ulmus glabra, were collected nationwide. Phytoplasma detection was performed by applying a universal 16Sr DNA-based quantitative PCR (qPCR) assay and a novel 'Ca. P. ulmi' specific qPCR assay targeting the 16S-23S spacer region. Both assays revealed that 28% of the samples were infected by 'Ca. P. ulmi', but infection rates of the elm species and regional incidences differed. The phytoplasma presence in the trees was not correlated to disease-specific symptoms. The survey identified a regional disparity of infection which was high in east, south and central Germany, whereas only a few infected sites were found in the western and northern parts of the country. Monitoring the seasonal titre of 'Ca. P. ulmi' in an infected tree by qPCR revealed a high colonisation in all parts of the tree throughout the year.
'Ca. P. ulmi' is widely present in elms in Germany. The rare occurrence of symptoms indicates either a high degree of tolerance in elm populations or a low virulence of pathogen strains enabling high infection rates in a long-living host.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In the last few years, the alarming spread of
Hymenoscyphus fraxineus
, the causal agent of ash dieback, has resulted in a substantial threat to native ash stands in central and northern Europe. ...Since leaves and leaf petioles are the primary infection sites, phyllosphere microorganisms are presumed to interact with the pathogen and are discussed as a source of biocontrol agents. We studied compound leaves from susceptible and visible infection-free trees in four ash stands with a high likelihood of infection to assess a possible variation in the bacterial microbiota, depending on the health status of the trees. The bacterial community was analyzed by culture-independent 16S rRNA gene amplicon sequencing and through the isolation and taxonomic classification of 2,589 isolates using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The bacterial community structure did not show significant differences. However, a set of amplicon sequence variants (ASVs) and MALDI groups belonging to
Luteimonas
,
Aureimonas
,
Pseudomonas
,
Bacillus
, and
Paenibacillus
were distinctly increased in tolerant trees, which may be associated with the ability of the tree to resist the pathogen. The most obvious differences were observed for
Luteimonas
, a genus that is also exclusively present in the healthy core microbiome. In a first
in vitro
screen of antagonists, approximately 11% of total isolates suppressed the growth of
H. fraxineus
, but a statistical test with two different
H. fraxineus
strains confirmed only the antagonistic activity of 8% of these isolates. The antagonistic isolates were assigned to
Bacillus velezensis
,
Pantoea vagans
, and
Pseudomonas caspiana.
Overall, our study provides a set of isolates or phylogenetic groups that might be involved in the process that prevents the penetration and spread of
H. fraxineus.
In the next step,
in planta
experiments are required with a longer period of exposure to
H. fraxineus
to evaluate effective isolates or consortia of isolates acting through direct antagonism or competition or indirectly by inducing resistance.
Phytoplasmas are insect-transmitted, uncultivable bacterial plant pathogens that cause diseases in hundreds of economically important plants. They represent a monophyletic group within the class ...Mollicutes (trivial name mycoplasmas) and are characterized by a small genome with a low GC content, and the lack of a firm cell wall. All mycoplasmas, including strains of 'Candidatus (Ca.) Phytoplasma asteris' and 'Ca. P. australiense', examined so far have circular chromosomes, as is the case for almost all walled bacteria.
Our work has shown that 'Ca. Phytoplasma mali', the causative agent of apple proliferation disease, has a linear chromosome. Linear chromosomes were also identified in the closely related provisional species 'Ca. P. pyri' and 'Ca. P. prunorum'. The chromosome of 'Ca. P. mali' strain AT is 601,943 bp in size and has a GC content of 21.4%. The chromosome is further characterized by large terminal inverted repeats and covalently closed hairpin ends. Analysis of the protein-coding genes revealed that glycolysis, the major energy-yielding pathway supposed for 'Ca. P. asteris', is incomplete in 'Ca. P. mali'. Due to the apparent lack of other metabolic pathways present in mycoplasmas, it is proposed that maltose and malate are utilized as carbon and energy sources. However, complete ATP-yielding pathways were not identified. 'Ca. P. mali' also differs from 'Ca. P. asteris' by a smaller genome, a lower GC content, a lower number of paralogous genes, fewer insertions of potential mobile DNA elements, and a strongly reduced number of ABC transporters for amino acids. In contrast, 'Ca. P. mali' has an extended set of genes for homologous recombination, excision repair and SOS response than 'Ca. P. asteris'.
The small linear chromosome with large terminal inverted repeats and covalently closed hairpin ends, the extremely low GC content and the limited metabolic capabilities reflect unique features of 'Ca. P. mali', not only within phytoplasmas, but all mycoplasmas. It is expected that the genome information obtained here will contribute to a better understanding of the reduced metabolism of phytoplasmas, their fastidious nutrition requirements that prevented axenic cultivation, and the mechanisms involved in pathogenicity.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In general, the definite determination of bacterial species is a tedious process and requires extensive manual labour. Novel technologies for bacterial detection and analysis can therefore help ...microbiologists in minimising their efforts in developing a number of microbiological applications.
We present a robust, standardized procedure for automated bacterial analysis that is based on the detection of patterns of protein masses by MALDI mass spectrometry. We particularly applied the approach for classifying and identifying strains in species of the genus Erwinia. Many species of this genus are associated with disastrous plant diseases such as fire blight. Using our experimental procedure, we created a general bacterial mass spectra database that currently contains 2800 entries of bacteria of different genera. This database will be steadily expanded. To support users with a feasible analytical method, we developed and tested comprehensive software tools that are demonstrated herein. Furthermore, to gain additional analytical accuracy and reliability in the analysis we used genotyping of single nucleotide polymorphisms by mass spectrometry to unambiguously determine closely related strains that are difficult to distinguish by only relying on protein mass pattern detection.
With the method for bacterial analysis, we could identify fire blight pathogens from a variety of biological sources. The method can be used for a number of additional bacterial genera. Moreover, the mass spectrometry approach presented allows the integration of data from different biological levels such as the genome and the proteome.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The γ-proteobacterium ‘Candidatus Arsenophonus phytopathogenicus’ is assigned as the major pathogen of “Syndrome des basses richesses”, a sugar beet disease characterised by a reduction in the sugar ...content of taproots and biomass yield. Despite the economic impact of this bacteriosis, diagnostics for this important pathogen currently rely on end-point PCR detection. Herein, we introduce a TaqMan qPCR for diagnostics of the agent targeting genes encoding a heat shock protein of the Hsp20 family and mannose-6-phosphate isomerase. Quantitation with synthetic oligonucleotides as standard showed that the developed TaqMan qPCR assays enable the detection of up to 100 target copies. A comparison between the TaqMan qPCR and end-point PCR for ‘Ca. A. phytopathogenicus’ detection was carried out on 78 sugar beet samples from different locations in southern Germany. The newly developed assays enable the fast, reliable and sensitive detection of ‘Ca. A. phytopathogenicus’ in sugar beet.
Sulfate-reducing bacteria (SRB) are key players of the carbon- and sulfur-cycles in the sediments of the world's oceans. Habitat relevant SRBs are often members of the Desulfosarcina-Desulfococcus ...clade belonging to the deltaproteobacterial family of Desulfobacteraceae. Despite this environmental recognition, their molecular (genome-based) physiology and their potential to contribute to organic carbon mineralization as well as to adapt to changing environmental conditions have been scarcely investigated. A metabolically versatile representative of this family is Desulfococcus multivorans that is able to completely oxidize (to CO
) a variety of organic acids, including fatty acids up to C
as well as aromatic compounds.
In this study the complete 4.46 Mbp and manually annotated genome of metabolically versatile Desulfococcus multivorans DSM 2059 is presented with particular emphasis on a proteomics-driven metabolic reconstruction. Proteomic profiling covered 17 substrate adaptation conditions (6 aromatic and 11 aliphatic compounds) and comprised 2D DIGE, shotgun proteomics and analysis of the membrane protein-enriched fractions. This comprehensive proteogenomic dataset allowed for reconstructing a metabolic network of degradation pathways and energy metabolism that consists of 170 proteins (154 detected; ~91 % coverage). Peripheral degradation routes feed via central benzoyl-CoA, (modified) β-oxidation or methylmalonyl-CoA pathways into the Wood-Ljungdahl pathway for complete oxidation of acetyl-CoA to CO
. Dissimilatory sulfate reduction is fueled by a complex electron transfer network composed of cytoplasmic components (e.g., electron transfer flavoproteins) and diverse membrane redox complexes (Dsr, Qmo, Hmc, Tmc, Qrc, Nuo and Rnf). Overall, a high degree of substrate-specific formation of catabolic enzymes was observed, while most complexes involved in electron transfer appeared to be constitutively formed.
A highly dynamic genome structure in combination with substrate-specifically formed catabolic subproteomes and a constitutive subproteome for energy metabolism and electron transfer appears to be a common trait of Desulfobacteraceae members.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The capacity to biomineralize is closely linked to the rapid expansion of animal life during the early Cambrian, with many skeletonized phyla first appearing in the fossil record at this time. The ...appearance of disparate molluscan forms during this period leaves open the possibility that shells evolved independently and in parallel in at least some groups. To test this proposition and gain insight into the evolution of structural genes that contribute to shell fabrication, we compared genes expressed in nacre (mother-of-pearl) forming cells in the mantle of the bivalve Pinctada maxima and the gastropod Haliotis asinina. Despite both species having highly lustrous nacre, we find extensive differences in these expressed gene sets. Following the removal of housekeeping genes, less than 10% of all gene clusters are shared between these molluscs, with some being conserved biomineralization genes that are also found in deuterostomes. These differences extend to secreted proteins that may localize to the organic shell matrix, with less than 15% of this secretome being shared. Despite these differences, H. asinina and P. maxima both secrete proteins with repetitive low-complexity domains (RLCDs). Pinctada maxima RLCD proteins-for example, the shematrins-are predominated by silk/fibroin-like domains, which are absent from the H. asinina data set. Comparisons of shematrin genes across three species of Pinctada indicate that this gene family has undergone extensive divergent evolution within pearl oysters. We also detect fundamental bivalve-gastropod differences in extracellular matrix proteins involved in mollusc-shell formation. Pinctada
maxima expresses a chitin synthase at high levels and several chitin deacetylation genes, whereas only one protein involved in chitin interactions is present in the H. asinina data set, suggesting that the organic matrix on which calcification proceeds differs fundamentally between these species. Large-scale differences in genes expressed in nacre-forming cells of Pinctada and Haliotis are compatible with the hypothesis that gastropod and bivalve nacre is the result of convergent evolution. The expression of novel biomineralizing RLCD proteins in each of these two molluscs and, interestingly, sea urchins suggests that the evolution of such structural proteins has occurred independently multiple times in the Metazoa.
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