Paraburkholderia and Herbaspirillum can promote the growth of Sorghum bicolor, but the underlying mechanisms are not yet known. In a pot experiment, sorghum plants grown on sterilized substrate were inoculated with Paraburkholderia tropica strain IAC/BECa 135 and Herbaspirillumfrisingense strain IAC/BECa 152 under phosphate-deficient conditions. These strains significantly increased Sorghum bicolor cultivar SRN-39 root and shoot biomass. Shotgun metagenomic analysis of the rhizosphere revealed successful colonization by both strains; however, the incidence of colonization was higher in plants inoculated with P. tropica strain IAC/BECa 135 than in those inoculated with H. frisingense strain IAC/BECa 152. Conversely, plants inoculated with H. frisingense strain IAC/BECa 152 showed the highest increase in biomass. Genomic analysis of the two inoculants implied a high degree of rhizosphere fitness of P. tropica strain IAC/BECa 135 through environmental signal processing, biofilm formation, and nutrient acquisition. Both genomes contained genes related to plant growth-promoting bacterial (PGPB) traits, including genes related to indole-3-acetate (IAA) synthesis, nitrogen fixation, nodulation, siderophore production, and phosphate solubilization, although the P. tropica strain IAC/BECa 135 genome contained a slightly more extensive repertoire. This study provides evidence that complementary mechanisms of growth promotion in Sorghum might occur, i.e., that P. tropica strain IAC/BECa 135 acts in the rhizosphere and increases the availability of nutrients, while H. frisingense strain IAC/BECa 152 influences plant hormone signaling. While the functional and taxonomic profiles of the rhizobiomes were similar in all treatments, significant differences in plant biomass were observed, indicating that the rhizobiome and the endophytic microbial community may play equally important roles in the complicated plant-microbial interplay underlying increased host plant growth.Bacteria from the genera  An essential step in plant–microbe interactions is the colonization of the rhizosphere by microorganisms. The rhizobiome is shaped by soil characteristics such as moisture, nutrient availability, texture, pH, and climate, but to an even greater extent by the host plant ,2,3,4,5.The first step in colonization is the migration of bacteria to the rhizosphere. To attract the desired microbes, the plant must exude substances that act as chemoattractants. The chemoattractant role of exudates was thought to be plant-specific , but rec2), this form of nitrogen is inaccessible to plants. N fixation by nitrogen-fixing microorganisms converts atmospheric N to ammonia, which is accessible to plants, via nitrogenase [2PO4) and dibasic (HPO42−) ions [3+, which tends to form insoluble hydroxides, making it inaccessible to plants [3 [Plant growth-promoting rhizobacteria (PGPR) are naturally occurring soil-borne bacteria that associate with plant roots and promote plant growth via multiple mechanisms. (i) Nitrogen (N) is the most vital nutrient for plant development. Although N is abundant in the atmosphere rogenase , P is no2−) ions . Upon ex2−) ions . P solub2−) ions . (iii) Po plants . Bacterio plants . Sideropo plants . (iv) Auo plants ,22,23,24o plants . (v) Etho plants . Some PGlants [3 . PGPR ablants [3 ,29.Herbaspirillum and Paraburkholderia  were idectively ,35,36,37romoters ,39. Incrromoters ,40,41. Rromoters ,42,43. IP. tropica strain IAC/BECa 135 and H. frisingense strain IAC/BECa 152 were previously isolated from sugarcane roots. Each bacterial strain inoculum was cultivated in dextrose, yeast, and glutamate (DYGS) liquid medium at 30 °C. The growth curve was determined, and the cells were harvested at exponential growth. The medium containing the cells was centrifuged , and genomic DNA was extracted using a Wizard Genomic DNA Purification Kit (Promega) according to the manufacturer’s instructions. The genomic DNA of each isolate was sequenced on a single PacBio P6C4 SMRT Cell , which was assembled using Prokka v1.11 [ka v1.11  and annoka v1.11  and the ka v1.11 ,47. CircSorghum bicolor, a pot experiment was carried out in a greenhouse over 30 days. Three treatments were applied to pots with autoclaved sandy substrate growing Sorghum bicolor cultivar SRN-39, namely, inoculation with P. tropica strain IAC/BECa 135, inoculation with H. frisingense strain IAC/BECa 152, and no inoculation (control). Each treatment contained six replicate pots.To evaluate the effects of the two bacterial inoculants on the growth of 3(PO4)2), which can be solubilized by microorganisms but cannot taken up directly by plants, was diluted in Hoagland nutrient solution and applied to the pots. Twenty-five milliliters of the nutrient solution was applied every two days.Seeds were disinfected as described by Liu et al. . After d8 cfu ml−1) [Bacterial isolates were taken from a single colony, grown on Petri dishes containing Luria–Bertani (LB) medium, incubated for 2–3 days at 30 °C, and stored at 4 °C. To prepare the bacterial inoculants, bacterial cultures were grown overnight at 31 °C in LB liquid medium, inoculated again in fresh LB medium, and allowed to grow until they reached the desired inoculum density (10fu ml−1) . A volumAfter four weeks, the plants were extracted from the pots, and rhizosphere soils were collected with a sterilized brush, immediately transferred to liquid nitrogen, and stored at −80 °C until DNA extraction. Subsequently, the root systems of the plants were washed with demi water, and the roots and shoots were separated.For root architecture measurements, the root systems were sectioned into three parts, spread along a rectangular acrylic tray, and placed in an EPSON scanner v.3.9.3 1NL. The measured root architecture parameters were specific root area , specific root length , average root diameter (AvD), and specific root density . All parameters were analyzed in the program WINRHIZOTM V2005b.After washing, the shoot and root parts were dried at room temperature for four hours until no remaining water was visible on the surface. The fresh weights of both parts were obtained using an electronic scale. The shoot and root parts were subsequently dried in an oven at 60 °C for 72 h. Biomass was calculated by dividing the dry weight by the fresh weight.From the six replicates per treatment, three replicates were randomly chosen for DNA extraction. DNA was extracted from 0.25 g of rhizosphere soil using a DNA Power-Soil DNA isolation kit . DNA integrity was checked by agarose gel (1.5%) electrophoresis in 1X Tris–EDTA (TBE) buffer. The gDNA was quantified using a Quant-iTTM dsDNA Assay Kit combined with Gen5 Data Analysis software . The total DNA was prepared as a MiSeq Illumina paired-end library and sequenced (3 replicates × 3 treatments = 9 metagenomes) using MiSeq technology.The bioinformatics steps were performed in a 64-node in-house cluster running Linux Ubuntu v16.04.4  with 1 TB of memory. The pipeline is available as a Snakemake v4.7.0 pipeline on request from the NIOO-KNAW GitLab. The workflow of the analyses is illustrated in Reads were preprocessed using Trimmomatic v0.36 . AdapterAfter quality control, reads were taxonomically classified using KrakenHLL v0.4 , a referFunctional profiles of the metagenomes were generated by assembling the reads using MEGAHIT v1.1.2  using thParaburkholderia tropica strain IAC/BECa 135 genome was deposited in National Center for Biotechnology Information (NCBI) with the accession number CP049134, and the Herbaspirillum frisingense strain IAC/BECa 152 genome was deposited in NCBI with the accession number CP049139. The metagenome sequences were deposited in the European Nucleotide Archive  under the accession number PRJEB36816 ENA.The To compare the taxonomic and functional profiles across treatments, normalization procedures were performed in R v3.4.0. Taxonomic abundances were normalized by multiplying the abundance by a sample-specific weight, which was calculated by dividing the number of matches by the total number of reads. Two normalization techniques were applied to the functional profiles, i.e., average genome size (AGS)  and openPotential bacterial interactions were determined by mapping the proteins predicted from the metagenomes to KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways that play an important role in the rhizosphere lifestyle : ko02010; carotenoid biosynthesis: ko00906; tryptophan metabolism: ko00380; phosphonate and phosphonate metabolism: ko00440; sulfur metabolism: ko00920; citrate cycle: ko00020; glyoxylate and dicarboxylate metabolism: ko00630; bacterial secretion systems: ko03070; QS: ko02024) using the KEGG Mapper—Reconstruct Pathway utility [P. tropica strain IAC/BECa 135 and H. frisingense strain IAC/BECa 152 genomes were compared against this database using DIAMOND v0.9.21 [A database containing protein sequences from proteins related to well-studied PGPR mechanisms, including siderophore production, phosphate solubilization, nitrogen fixation, nodulation, plant hormone production, nitrification, and secretion systems, was manually compiled using the public resources UniProt  and KEGG v0.9.21   identifiers of the annotated proteins from the bacterial genomes to the following KEGG pathways: tryptophan metabolism , phenylalanine metabolism , cysteine and methionine metabolism (ACC-deaminase and ethylene), alpha-linolenic acid metabolism , zeatin biosynthesis (cytokinin), carotenoid biosynthesis , and diterpenoid biosynthesis .To analyze the capacities of P. tropica strain IAC/BECa 135 and H. frisingense strain IAC/BECa 152 were mapped to the KEGG ABC transporter pathway (identifier ko02010) using the KEGG Mapper—Reconstruct Pathway utility.To compare the diversity of ABC transporters, the KO identifiers of the annotated proteins from the genomes of P. tropica strain IAC/BECa 135 and H. frisingense strain IAC/BECa 152 isolates were assembled and annotated  resulted in 25,807,210 reads (6219.3 Mb) with a mean length of 241 bp  were generated from nine rhizosphere shotgun metagenomics samples (3 × 3 replicates) of the f 241 bp .p = 0.000262). The bacterial community differences between the treatments were almost entirely attributable to the inoculants  of the reads per sample could be classified as bacteria at the genus level, with a range of 36% to 87% A. The proculants . We obseP. tropica strain IAC/BECa 135 inoculant metagenomes were taxonomically classified, the assembly quality of these metagenomes was underwhelming compared to the other metagenomes  and average contig size were strongly correlated (Pearson correlation coefficient r = 0.62), whereas the correlation between the average contig size and percentage of annotated genes was low (r = 0.27), implying that the quality of the metagenome assembly did not strongly influence the annotation process.Although most of the reads from the agenomes . The metP. tropica strain IAC/BECa 135 metagenomes.The functional profiles of the metagenomes were highly similar at a high level of abstraction. The distribution of COG terms among the metagenomes did not differ by more than 1% between treatments . AdditioP. tropica strain IAC/BECa 135 and H. frisingense strain IAC/BECa 152 genomes were compared according to the presence or absence of PGPR gene homologues. The presence of genes related to indole-3-acetate (IAA) synthesis, nitrogen fixation, nodulation, siderophore production, and phosphate solubilization in both genomes underlined the genetic capacity of both strains to promote plant growth. PGPR genes are present in both genomes, however, P. tropica strain IAC/BECa 135 possesses more genes related to siderophore production, specifically, genes similar to those responsible for pyoverdine synthesis  identifiers of the annotated genes from the  systems . P. trop systems . The typnization . Again, BECa 152 . The monH. frisingense strain IAC/BECa 152 treatments were most similar (87.6% shared KOs), consistent with the taxonomic profiles. Between 43% and 49% of the predicted genes were annotated. The unique KOs for each treatment were mapped to KEGG pathways to identify modules present in the metagenome of only one treatment. The KEGG categories with the largest numbers of unique KOs across treatments were metabolism and environmental signal processing. No complete biosynthetic modules unique to a metagenome of a single treatment were found.When the KOs were used as a measure of functionality, the functional capacities of the microbial communities assembled in the rhizosphere by the inoculation of the two strains differed by only a few percent D. The fuSorghum bicolor rhizosphere after inoculation with P. tropica strain IAC/BECa 135 and H. frisingense strain IAC/BECa 152 under phosphate-deficient conditions. Inoculation of these bacteria in Sorghum bicolor cultivar SRN-39 led to significant increases in root and shoot biomass. Although P. tropica strain IAC/BECa 135 was much more abundant in the rhizosphere than H. frisingense strain IAC/BECa 152, plants treated with H. frisingense strain IAC/BECa 152 demonstrated higher root and shoot biomass. However, the current study was carried out in sterilized substrate conditions; in field conditions, the responses of these strains might be different due to expected microbiome competition in rhizosphere.This study was the first to analyze and compare the community composition in the P. tropica in Triticum aestivum (wheat), tomato, and microalgae (Chlorella sp.) via nitrogen fixation, phosphate solubilization, and phytohormone production [P. tropica strain IAC/BECa 135.Previous studies showed growth-promoting effects of oduction ,69, but H. frisingense was demonstrated in sugarcane [Miscanthus sinensis (both C4 grass species). However, in M. sinensis, the effect was due not to increased nutrient levels but to an influence of this endophytic bacterium on plant hormone signaling [Herbaspirillum species confirmed the presence of many genes involved in plant hormone synthesis in the H. frisingense genome [H. frisingense strain IAC/BECa 152 were present, as previous research showed that the mode of action of H. frisingense primarily involves the intercellular apoplastic space. Genomic analysis of H. frisingense strain IAC/BECa 152 showed the presence of genes involved in IAA synthesis and ACC-deaminase, as well as genes involved in direct PGPR traits such as nitrogen fixation and phosphate solubilization; however, the activity of these genes could not be quantified, as the transcriptome was not measured. Despite the lack of phytohormone synthesis genes, endophytic microbes were shown to influence hormone signaling by synthesizing hormone mimics and effector proteins that influence hormone production in planta [H. frisingense strain IAC/BECa 152 but were not detected by the homology-based approach used in this study. In addition, the presence of genes involved in terpenes and other antimicrobial compounds could be involved in plant defenses against bacterial, fungal, and viral pathogens [Growth promotion by ugarcane , sorghumugarcane , and Misignaling . A studye genome . In thisn planta . This asathogens .P. tropica strain IAC/BECa 135 genome is enriched in ABC transporters, biosynthetic clusters encoding enzymes involved in polysaccharide synthesis, QS systems, and secretion systems compared to the H. frisingense strain IAC/BECa 135 genome. These genetic features are involved in the colonization process and could thus explain the extensive rhizosphere colonization by P. tropica strain IAC/BECa 135. Another plant-beneficial Burkholderia strain, Burkholderia australis , was shown to form a biofilm on sugarcane (a close relative of sorghum) roots, in which the expression of EPS genes, cytochrome genes, and energy pathway genes were upregulated, lipopolysaccharide (LPS) and flagella biosynthesis (which are often recognized as virulence factors by plants) gene expression were downregulated, and additional energy pathways were activated [B. australis, whereas P. tropica strain IAC/BECa 135 root colonization appears to be dependent on the orobanchol concentration in Sorghum bicolor root exudates [P. tropica strain IAC/BECa 135 could enable the bacterium to integrate many signals and coordinate gene expression accordingly. Furthermore, the products of variable biosynthetic gene clusters can play a role in biocontrol activity against phytophatogens, such as fungi, bacteria, and nematodes, and induce plant biomass increase, as demonstrated for other Burkholderia and Paraburkholderia species. In addition, the large number of distinct ABC transporters may enable P. tropica strain IAC/BECa 135 to sense many environmental stimuli, thus conferring a colonization advantage. The genomic differences between P. tropica strain IAC/BECa 135 and H. frisingense strain IAC/BECa 152 may reflect the differences between the rhizospheric and endophytic lifestyles, and further studies on gene expression may confirm the endophytic colonization of S. bicolor by H. frisingense strain IAC/BECa 152.The ctivated . Interesexudates , indicatP. tropica strain IAC/BECa 135 was more successful in colonizing the rhizosphere of Sorghum bicolor cultivar SRN-39 than H. frisingense strain IAC/BECa 152. This advantage was partially explained by the P. tropica strain IAC/BECa 135 genome, which is enriched in features important for colonization. As inoculation with H. frisingense strain IAC/BECa 152 led to a greater increase in biomass than inoculation with P. tropica strain IAC/BECa 135 despite a lower degree of rhizosphere colonization, it is possible that a high concentration of H. frisingense strain IAC/BECa 152 was present in the intercellular apoplastic space in the roots. The mechanisms by which P. tropica strain IAC/BECa 135 and H. frisingense strain IAC/BECa 152 promote plant growth might be complementary, i.e., H. frisingense strain IAC/BECa 152 seems to influence plant hormone regulation, whereas P. tropica strain IAC/BECa 135 comprehensively colonizes the root system and surrounds the roots with biofilm, protecting the roots against pathogens and increasing nutrient availability through a range of mechanisms. While the functional and taxonomic profiles of the rhizobiomes of all treatments were similar, significant differences in biomass were observed, indicating that the rhizobiome and endophytic microbial community may play equally important roles in the complicated plant–microbial interplay underlying the increased growth of the host plant.In summary, as illustrated in P. tropica strain IAC/BECa 135 and H. frisingense strain IAC/BECa 152 presumably promote growth through different mechanisms, co-inoculation of these bacteria should lead to greater increases in root and shoot biomass than inoculation of a single bacterium. This hypothesis could be tested by performing an experiment similar to this study, in which P. tropica strain IAC/BECa 135 and H. frisingense strain IAC/BECa 152 are inoculated at different concentrations to find an optimal inoculum.Finally, as


Davidia involucrata Baill. is a kind of tertiary paleotropical plant floristic relic species unique to China. This rare plant is disappearing due to poor adaptability and serious poaching. We first assembled the complete chloroplast (cp) genome of Davidia involucrata Baill. by Illumina paired-end reads data. The whole genome was 169,085 bp, consisting of a pair of inverted repeats of 169,379 bp, large single copy region and a small single copy region . The cp genome contained 90 genes, including 64 protein-coding genes, 22 trRNA genes and 4 rRNA genes. The overall GC content of the whole genome was 38.04%. A neighbor-joining phylogenetic analysis demonstrated a close relationship between Davidia involucrata Baill. and Nyssa yunnanensis. Davidia involucrata is a kind of tertiary paleotropical plant floristic relic species unique to China. Davidia involucrata is also named as Chinese dove tree, because the lower bud looks like a white dove ready to fly and has a high ornamental and research value . A voucher specimen (JN20195052) was deposited in the Herbarium of the Plant Biotechnology Center of Jilin Agricultural University, Changchun, China.In this study, Davidia involucrata Baill. (GenBank: NC_031376.1) from high-throughput sequencing data. The existing chloroplast Genome sequence of ginkgo biloba was downloaded from the National Center for Biotechnology Information’s Organelle Genome Resources database (nc_031376.1) as the reference sequence, and the chloroplast Genome of ginkgo biloba was assembled using SPAdes v3.6.0 software  with default parameters, and the genes were functionally annotated by combining with NR (http://www.ncbi.nlm.nih.gov/) database  was used to record rRNA for comments. We used Homblocks  region of 96,712 bp and a small single-copy(SSC) region of 67,667 bp, separated by two inverted repeat regions (IRs) of 2353 bp. It contained 90 genes, including 64 protein-coding genes, 4 ribosomal RNA genes, and 22 tRNA genes. The phylogenetic tree reveals that all the species of Nyssaceae formed a monophyletic clade with high-resolution value in cornales and Nyssa yunnanensis W.C.Yin is closely related with Davidia involucrata Baill. (The complete cp-DNA of a Baill. .


Eidophasiaassmannisp. nov., a new species of Plutellidae from the alpine zone of Russian Altai Mountains, is described from diagnostic morphology and DNA barcodes. Male adult and genitalia are illustrated, whereas the female sex remains unknown. The species inhabits alpine scree with patchy herbaceous plants and is considered as possible endemic species of the Altai Mountains. An updated checklist of the 13 global Eidophasia Stephens, 1842 species is provided. The likely polyphyly of the genus is discussed from molecular data of the barcode region of the mt COI gene. Lepidoptera fauna from the Altai region attracted attention from lepidopterists early on, e.g., The Plutellidae from the Republic of Altai is described. Plutellidae are a moderately diverse family of Yponomeutoidea with ca. 150 described species from 48 genera listed on a worldwide scale , and with few taxa added more recently , being more widely distributed in the southern parts of the country  to obtain DNA barcodes using the standard high-throughput protocol described in BOLD. Obtained sequences range between 522 and 658 bp, with 88 specimens represented by a full DNA barcode. The sequenced material includes 41 specimens of Eidophasia, whereas the remaining 59 sequences depict the type species of the outgroup genera Plutella, Plutelloptera, Pseudoplutella Baraniak, 2007, and Rhigognostis Staudinger, 1857. Furthermore, an unpublished specimen of E.albidorsella  (specimen ID USNMENT00657823) was considered for analysis. Sequences were submitted to GenBank, and further details including complete voucher data and images can be accessed in the public dataset “DS-EIDOASMA Eidophasiaassmanni sp.n.” https://dx.doi.org/10.5883/DS-EIDOASMA in BOLD. Degrees of intra- and interspecific variation of DNA barcode fragments were calculated under Kimura 2 parameter model of nucleotide substitution using analytical tools of BOLD systems v. 4.0. A Neighbour-joining tree of DNA barcode data of central and south-eastern European taxa was constructed using MEGA 6  system as implemented on BOLD (OTUs) and automatically assigns new sequences, irrespective of their previous taxonomy and origin. Similarities or differences between BINs and morphological species identification were assessed.Identification success was furthermore assessed by the Barcode Index Number   . Its typrd segment equal or slightly longer than the 2nd segment, and the antennae partially knobby, due to specialised scales. However, as discussed earlier by Eidophasia to date.The generic definition followed in this paper is largely based on E.dammersi , E.albidorsella , E.vanella ), and one to New Guinea . E.zukowskyi Amsel, 1939 and E.infuscata Staudinger, 1870 have been upgraded to species level .The genus is mainly Holarctic with currently 13 known species. Three species are restricted to North America Spania Guenée, 1845Hufnagelia Reutti, 1853Eudophasia Herrich-Schäffer, 1853 (misspelling)messingiella species groupThe Eidophasiamessingiella  (Plutella)transversella = muellerella Rougemont, 1903= aereolella Lhomme, 1949= Eidophasiainfuscata Staudinger, 1870Eidophasiatauricella Staudinger, 1880Eidophasiaalbifasciata Issiki, 1931Eidophasiadammersi  (Plutella)Eidophasiaalbidorsella  (Plutella)Eidophasiavanella  (Plutella)Eidophasaassmanni sp. nov.syenitella species groupThe Eidophasiasyenitella Herrich-Schäffer, 1854 (Eudophasia [sic])concinnella Christoph, 1888= Eidophasiazukowskyi Amsel, 1939Eidophasiahufnagelii  (Plutella)Eidophasiainsulella  (Caunaca)Eidophasiaperistigma Diakonoff, 1955Taxon classificationAnimaliaLepidopteraPlutellidae2B328278-3597-5B17-9B16-F542E129FEA3http://zoobank.org/A204788A-24C2-41A3-8F06-6DABDD5DDF72Holotype ♂: “Russia, Altai Republic, / Ulagan distr., 10 km NE / Aktash vill., Kuraj Mts. / Range, between rivers / Korumdyajry and Yarlyamry, / 50°20'N, 87°45'E, stone / tundra, 2750–2800 m, / 07.08.2016, leg. Huemer & / Wiesmair, TLMF 2016–020” “DNA Barcode / TLMF Lep 21215” “YPO 162 ♂ P. Huemer” . Paratype: 1 ♂, same data as holotype, but 2900–3000 m, 30.vii.2016, DNA Barcode TLMF Lep 20484 .Eidophasiaassmanni is unmistakable in habitus due to the inconspicuous wing markings, which are clear and prominent in E.assmanni, and white or yellow in all other known species of the genus. The male genitalia are unique in Eidophasia by the oblong shape of the valva with straight dorsal and ventral edges and particularly the very long and apically pointed sacculus with largely reduced spiniform setae on distal end. In E.messingiella and related taxa the valva is obovate with a curved sacculus and sets of spiniform setae at apex , the minimum distance to the Nearest BIN in BOLD, E.vanella, is 5.25% (p-distance), whereas the Nearest Neighbour in our dataset is a specimen of E.messingiella with 5.88% divergence .th birthday and in recognition of his particular support of Natural History Collections already in his former and present career.The species is dedicated to the Director of Tyrolean Federal State Museums Mag. Dr. Peter Assmann to his 55Eidophasia  and E.syenitella .The interspecific divergence of  9% Fig. . This exPlutella and its allied genera need further assessment. This is particularly relevant for the genus Eidophasia which in its current scope is most likely polyphyletic as indicated by its heterogeneous morphology (Eidophasia were divided into two clusters. One cluster (the messingiella species group) included the type species of the genus, E.messingiella, and three congeners: E.vanella, E.albidorsella, and E.assmanni. The other cluster (the syenitella species group) included two species of Eidophasia, E.syenitella and E.hufnagelii. The former cluster shows two major differences in genital features from the latter: the distal part of sacculus of the valva flapped in various degrees, and the ductus bursae entirely membranous except the antrum. The wing patterns and the genital features of E.insulella and E.peristigma suggest that they also belong to the syenitella species group. The syenitella species group may represent a separate genus from Eidophasia but their generic assignment is pending in this study.As already discussed by rphology  and the E.assmanni clearly show that it belongs to the messingiella species group. The messingiella species group exhibits considerable interspecific barcode divergence which should be further assessed in the future in an integrative taxonomic study on a worldwide scale.The DNA barcode sequences of


Yersinia pestis minimum infection prevalence in fleas collected from Tamias spp. chipmunks in the Sierra Nevadas  during 2013–2015. Y. pestis–positive fleas were detected only in 2015 (year of plague epizootic), mostly in T. speciosus chipmunks at high-elevation sites. Plague surveillance should include testing vectors for Y. pestis.We describe  Yersinia pestis, the bacterium that causes plague, is carried by multiple flea species in western North America, where sciurids are often the primary reservoirs  was the host that was most frequently seropositive  and lodgepole (T. speciosus) chipmunks in Yosemite National Park and surrounding areas during 2013–2015. We focused on T. speciosus chipmunks because of their documented role in the 2015 epizootic .T. alpinus chipmunks and 538 pools  from 389 T. speciosus chipmunks (Y. pestis DNA in flea pools (We identified key flea specimens (N = 122) . All 3 of the flea species  most commonly found on T. speciosus and T. alpinus chipmunks were found to be positive for Y. pestis . Unfortunately, these fleas were too damaged to identify morphologically, and molecular species identification was not possible.We found surveyed  Table 2.. pestis  Table 1 ive flea  Table 2.Y. pestis–positive flea pools were detected at 5 of 6 high-elevation  study sites in 2015. Many of these sites are areas of high human activity, with popular hiking trails or established campgrounds. In 2015, plague risk assessments, including testing flea pools and rodent carcasses for Y. pestis DNA and rodent serology, also took place at lower elevation sites  in and around the park; these surveys detected Y. pestis at 4 of 17 locations (Y. pestis prevalence in fleas during a plague epizootic year in California. Our results support integrating flea testing, especially those at high-elevation sites, into regular surveillance.Altogether, our data indicate a dramatic shift in More information about plague epizootic dynamics in chipmunk fleas, Sierra Nevada, California, USA, 2013–2015.


Triticum spp.) has been an important staple food crop for mankind since the beginning of agriculture. The genus Triticum L. is composed of diploid, tetraploid, and hexaploid species, majority of which have not yet been discriminated clearly, and hence their phylogeny and classification remain unresolved. Genotyping-by-sequencing (GBS) is an easy and affordable method that allows us to generate genome-wide single nucleotide polymorphism (SNP) markers. In this study, we used GBS to obtain SNPs covering all seven chromosomes from 283 accessions of Triticum-related genera. After filtering low-quality and redundant SNPs based on haplotype information, the GBS assay provided 14,188 high-quality SNPs that were distributed across the A (71%), B (26%), and D (2.4%) genomes. Cluster analysis and discriminant analysis of principal components (DAPC) allowed us to distinguish six distinct groups that matched well with Triticum species complexity. We constructed a Bayesian phylogenetic tree using 14,188 SNPs, in which 17 Triticum species and subspecies were discriminated. Dendrogram analysis revealed that the polyploid wheat species could be divided into groups according to the presence of A, B, D, and G genomes with strong nodal support and provided new insight into the evolution of spelt wheat. A total of 2,692 species-specific SNPs were identified to discriminate the common (T. aestivum) and durum (T. turgidum) wheat cultivar and landraces. In principal component analysis grouping, the two wheat species formed individual clusters and the SNPs were able to distinguish up to nine groups of 10 subspecies. This study demonstrated that GBS-derived SNPs could be used efficiently in genebank management to classify Triticum species and subspecies that are very difficult to distinguish by their morphological characters.Wheat ( Triticum spp.) is the most widely cultivated food crop worldwide. The large amount of wheat accessions in the world’s genebanks has reflects the importance of wheat as a world crop  basic genomes . The majpulation . Variouspulation . Landjevpulation .Brassica spp. (The efficient introgression of novel genes from wild relatives to cultivar genotypes greatly depends on accurate identification of the wild species. Despite having standard procedures in genebanks such as the genebank management system (GMS) for efficient management of plant genetic resources, incorrect classification is not uncommon. Misidentification of species has been reported in crops, including rice , yams G, Brassicica spp. , and alsica spp. . Variousica spp. . Speciesica spp. , which hica spp. .T. aestivum) and their progenitor durum wheat (T. turgidum) are almost indistinguishable morphologically, making classification difficult. Different genes from nuclear and chloroplast genomes are utilized to identify the phylogenetic relationships existing between plant species including wheat  germplasm collection and National Agrobiodiversity Center at the National Institute of Agricultural Sciences, Rural Development Administration (RDA), Republic of Korea  cultivars and landraces were specifically used to find species-specific SNPs markers. Fresh leaf tissue was harvested from 3-week-old germinated seedling and total DNA was extracted using the DNeasy® Plant Mini kit  according to the manufacturer’s instructions.We collected a total 283 accessions representing 17 of Korea . Among tPstI (CTGCAG) and MspI (CCGG) at 37°C for 3 h and then the GBS libraries were constructed according to the protocols described previously  with Synergy HTX Multi-Mode Reader . The normalized DNA was codigested with the restriction enzymes (New England Biolabs) eviously . After r® Gel using the PCR Clean-up Kit . The purified samples were polymerase chain reaction (PCR) amplified with 25 pmol of primer  using Bowtie2  to correct misalignments due to the presence of indels  . The HapK with default parameters, which retains all principal components (PCs). To determine the optimal number of PCs to retain in the discriminant analysis, we used the cross-validation function  to confirm the correct number of PCs to be retained  identified K = 5 and eight as the most suitable modeling choices.To investigate the population structure, admixture analysis was performed on 114 individuals using the ADMIXTURE tool3 . The admTriticum genotypes were estimated with the modified All SNPs were concatenated into a single alignment. PAUP 4.0b10 was used to calculate score for the substitution of SNPs and Bayesian analyses were conducted with the GTR + G nucleotide substitution model using MrBayes v. 3.2.6. The GTR + G model was chosen in both AIC and hLRTs models for the model estimation. The model was estimated by MrModelTest v. 2.4 using the calculated score as the input value . The treT. aestivum/T. turgidum accession at the species level. PCoA of genetic variation among species groups was performed using GCTA software  was performed to characterize the genotypes between common and durum wheat, which classified software .Illumina NextSeq500 generated a total of 1,259,242,576 raw single-end sequence reads from seven GBS runs for 283 samples . The numTriticum species discrimination, which includes 114 accessions. After filtering for missing values, physical distance, and minor allele frequency, a total of 14,188 final SNPs were distributed among the seven chromosomes  was plotted against ΔK, which showed a sharp peak at K = 5 or K = 8  were clustered individually in cluster 4. Similarly, the fewer accessions of T. monococcum, T. timopheevii, and T. urartu were present in cluster 3, 5, and 6, respectively. As expected, T. zhukovskyi accession was found in cluster 5 along with T. timopheevii. Distribution of molecular variance among and within population clusters was estimated using AMOVA. Results revealed that based on pairwise PhiPT values, the genetic variability between (67%) clusters was greater than the variability within (33%) clusters , T. urartu (AA genome), T. turgidum (AABB genome), T. timopheevii (AAGG genome), T. aestivum (AABBDD genome), and T. zhukovskyi (AAAAGG genome). The ML tree of an individual genome such as the A, B, and D showed a highly resolved phylogeny with high bootstrap support  is clustered more closely with the AABB(DD) lineage. All the Triticum species such as T. aestivum, T. turgidum, T. monococcum, T. timopheevii, T. urartu, and T. zhukovskyi were identified successfully. However, the individual genomes failed to differentiate the Triticum subspecies successfully on the ML tree of the A, B, and D genome. Hence, the Bayesian phylogenetic tree for all the 114 accessions along with Aegilops accessions was constructed for a better visualization of their relationships. The Bayesian phylogenetic reconstruction of Triticum species showed a highly resolved phylogeny with higher >50% nodal support  (AABBDD) was located on two different clades, with four European accessions tied to T. turgidum (AABB) and two Asian to T. aestivum (AABBDD) clade, suggested that the evolution of European and Asian spelt wheat may be different.A total of 114  support . The ML  support . In the DMIXTURE . The phy genomes . HoweverT. aestivum (AABBDD genome) and its wild progenitor T. turgidum (AABB genome) was found to be difficult to distinguish because of their paraphyletic relationship. Initially, a total of 8,269 fine SNPs were filtered from the raw variants to discriminate the common/durum wheat cultivar and landraces. Further, in Pearson’s chi-square test, a total of 2,692 species-specific SNPs were detected at each level. In PCoA grouping, the two wheat species formed individual clusters and the SNPs were able to divide up to 9 groups of 10 subspecies  and durum wheat  accessions were detected and varied from three on chromosome 4D to 86 on chromosome 5A  accessions and a second composed exclusively of durum wheat  accessions.In the first step (node 1), a total of 776 species-specific SNPs between the common  and the FTPC holds 43.7 and 27.6% of total variability (In the second step (node 2), we searched species-specific SNPs that discriminate subspecies within common and durum wheat accessions. We found 15.4 and 8.5% of total variability in FTPCs of 2A . A totalCs of 2T . In thisCs of 2T  discrimi266 SNPs  in 2–2T iability .T. aestivum subsp. spelta from other two subspecies and a total of 166 SNPs , 11 and 10.5% of total variability in FTPC of 4A were detected , a total of 275 SNPs  in 3A wi166 SNPs  in 3T wi166 SNPs  discrimidetected  with 37 detected . The SNP 24 SNPs  in 4T wi 24 SNPs  differenTriticum complex by developing genome-wide SNP datasets. As expected, GBS identified abundant genome-wide SNPs, with varying missing levels. The missing SNP datasets have been reported as a central drawback in low coverage genome sequencing such as Diversity Array Technology (DArT), GBS, etc. . Similarly, the identification of a relatively higher frequency of SNPs showing transition substitutions (62.3%) than transversions is consistent with previous genome-wide SNP discovery studies in crop plants, including wheat . The possible explanation for high variation between clusters is the inclusion of different wheat genotypes. According to T. aestivum and its wild progenitor T. turgidum genotypes presenting in the respective clusters. The largest PhiPT value was observed between clusters 1 (T. aestivum) and 3 (T. monococcum), indicating genetic variability between these two species is the greatest and genetic structure is most different. In the lineage of common wheat, it is well known that the A genome originated from T. urartu. There was no sharing genome between T. monococcum and T. aestivum in the wheat hybridization process from diploid to hexaploid, resulting in distinct genetic variability between these two species. Moreover, the results showed all the T. turgidum subsp. carthlicum accessions were found in the T. aestivum cluster (cluster 1).AMOVA analysis results on the basis of the wheat genotypes indicated a higher genetic variation between (67%) rather than within (33%) clusters. These variations were significant according to the partitioning value  were clustered individually in cluster 4. Meanwhile, the pairwise PhiPT value (0.078) indicates slightly higher variation within the subspecies of T. turgidum (cluster 4/cluster 2) when compared with the two wheat species (0.055) of T. aestivum/T. turgidum (cluster 1/cluster 2). Generally, there are more cultivars and landraces in common and durum wheat, which are used mainly for cultivation and breeding programs. Other T. turgidum subspecies, on the other hand, are not often used. This may be one reason that the genetic diversity of these species is maintained. species . Similaroccoides , which cTriticum species and subspecies except T. timopheevii and T. zhukovskyi. Though the T. timopheevii and T. zhukovskyi accessions formed an individual (cluster 5) cluster representing their genomic differences from other species, the present study has some limitation owing to lack of complete genomic information of the G genome. As sequence read alignment to the reference genome is a fundamental step in genomic studies , T. urartu (AA genome), T. turgidum (AABB genome), T. timopheevii (AAGG genome), T. aestivum (AABBDD genome), and T. zhukovskyi (AAAAGG genome). Of these species, T. urartu exists only in its wild form, whereas T. aestivum and T. zhukovskyi exist only as cultivated forms. The other species, T. monococcum, T. turgidum, and T. timopheevii, have both a wild and a domesticated form  was hybridized with Ae. speltoides Tausch (SS genome) in the initial stage of the wheat hybridization event, resulting in the genesis of wild emmer wheat (T. turgidum subsp. dicoccoides). Similarly, T. aestivum and its wild progenitor T. turgidum genotypes were clustered closer to each other. T. aestivum (AABBDD genome) is thought to have arisen through hybridization of T. turgidum with the wild wheat species Ae. tauschii Coss. (DD genome) (T. timopheevii accessions were clustered separately next to T. urartu as the tetraploid T. timopheevii (AAGG genome) species are believed to have evolved less than 0.5 million years ago through hybridization between T. urartu and a species that belonged to the lineage of the current wild wheat species, Ae. speltoides Tausch (SS genome) (T. zhukovskyi (AAAAGG genome) accession was clustered together with T. timopheevii (AAGG genome) accessions. T. zhukovskyi originated through hybridization of T. timopheevii with the cultivated einkorn T. monococcum in the Transcaucasus. Our molecular study based on the GBS-derived SNPs demonstrated that the T. timopheevii lineage is closer to the AABB(DD) lineage than the AA genome species on the basis of the B genome tree, in which T. timopheevii was clustered with T. aestivum species before T. urartu, although there is no B genome in both T. timopheevii and T. urartu (Ae. speltoides-like (SS genome) species, which is believed as a common progenitor for the B and G genomes of T. turgidum and T. timopheevii, respectively, and these genomes can recombine at a frequency of 30% (The diploid AA genome lineages ears ago . In the  genome) . Convers genome) . As expe. urartu . This rey of 30% .Triticum subsp. spelta (AABBDD) was clustered in two different positions showing polyphylesis in the Bayesian tree and also in the ML tree of the A and B genomes  and emmer wheat (T. turgidum subsp. dicoccon), resulting in the diversification of the A and B genomes (T. turgidum subsp. dicoccon) and the polyphyletic nature of spelt wheat, as mentioned in previous reports.More interestingly, the  genomes . There a genomes . The dis genomes  would al genomes , 2004. S genomes . Our resTriticum species, which could be misclassified or misidentified accessions. Similarly, based on GBS-derived SNPs, T. monococcum and T. urartu accessions were found to be misclassified in the genebank management system, which needs to be considered for further evaluation. Recently, The Bayesian phylogenetic tree, however, showed that some durum wheat accessions were clustered with other T. aestivum and T. turgidum species  or three genomes ; however, they were reported as very closely related species with only a low level of sequence diversity. It is generally accepted that the synthetic wheat T. aestivum (AABBDD genome) was derived through intergeneric hybridizations that occurred between species of Triticum and Aegilops  genotypes could not be classified efficiently, as they were found closer to common wheat (T. aestivum) accessions. However, in this study, we identified a total of 2,692 species specific SNPs  and durum (T. turgidum) wheat species and subspecies, as expected. DAPC and population structure analysis revealed that there are six groups that can be further classified based on their genotypes . The Bayesian and maximum likelihood (ML) phylogenetic reconstruction of Triticum species and subspecies showed a highly resolved phylogeny, with more than 50% nodal support. The dendrogram revealed the polyploid wheat species could be divided into groups according to the presence of A, B, D, and G genomes. A total of 2,692 GBS-derived SNPs were able to identify the hexaploid wheat from their wild relatives, which will help with accurate classifications of genebank accession. The study has proved that the GBS is a useful and reliable tool for the identification of high-quality SNPs, which clearly enhanced the phylogenetic resolution of the Triticum species. This study could be a first step toward genome-wide mapping for the identification of SNP markers in plants having complex genomes with numerous species and subspecies. Finally, this study also allowed us to identify a few misclassified accessions from the genebank collection, where the majority of wheat accessions are identified in an individual cluster. Further development of species-specific SNP-based barcodes could be useful for rapid and precise identification of germplasm resources for the wheat breeding program.In summary, we collected a total of 283 PRJNA601245, https://www.ncbi.nlm.nih.gov/bioproject/PRJNA601245.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI, Accession number DH conceived and designed the experiments. RS, G-AL, and KL performed the experiments and analyzed the data. M-JS and SK conducted project coordination and analysis. J-RL and G-TC contributed materials. RS and KL drafted the manuscript and figures. DH reviewed and edited the final draft. All authors contributed to the revision of the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Phytophthora sansomeana infects soybean and causes root rot. It was recently separated from the species complex P. megasperma sensu lato. In this study, we sequenced and annotated its complete mitochondrial genome and compared it to that of nine other Phytophthora species. The genome was assembled into a circular molecule of 39,618 bp with a 22.03% G+C content. Forty-two protein coding genes, 25 tRNA genes and two rRNA genes were annotated in this genome. The protein coding genes include 14 genes in the respiratory complexes, four ATP synthase genes, 16 ribosomal proteins genes, a tatC translocase gene, six conserved ORFs and a unique orf402. The tRNA genes encode tRNAs for 19 amino acids. Comparison among mitochondrial genomes of 10 Phytophthora species revealed three inversions, each covering multiple genes. These genomes were conserved in gene content with few exceptions. A 3' truncated atp9 gene was found in P. nicotianae. All 10 Phytophthora species, as well as other oomycetes and stramenopiles, lacked tRNA genes for threonine in their mitochondria. Phylogenomic analysis using the mitochondrial genomes supported or enhanced previous findings of the phylogeny of Phytophthora spp. Phytophthora includes many devastating pathogens infecting economically important crops [P. infestans, the pathogen that causes potato late blight and the culprit behind the Irish potato famine [Phytophthora genus has expanded significantly. With approximately 55 species described in 1999, it expanded to 105 species by 2007 [Phytophthora is a genus in the Peronosporales of oomycetes. Oomycetes produce hyphae and are morphologically similar to fungi. However, they are phylogenetically distant. Oomycetes belong to the major group Stramenopila, which also include diatoms and brown algae [The genus nt crops . Perhapso famine . During  by 2007  and 117  by 2007 . Currentwn algae –10.Phytophthora species into 10 clades [P. sansomeana is one of the recently described species in clade 8 [P. megasperma sensu lato, it is morphologically similar to but phylogenetically distinct from P. megasperma sensu strictu. P. sansomeana was isolated from soybean in Indiana in 1990 [P. sojae, is usually absent in P. sansomeana-infected soybean plants. This pathogen overwinters as oospore in the soil and in soybean debris. Also included in this species are isolates from Douglas-fir and alfalfa [Phylogenetic analysis grouped 0 clades , 11, 12. clade 8 . Previou in 1990  and has  in 1990 , Canada  in 1990 , 17. It  alfalfa .Phytophthora has been increasing due to its economic impact. Molecular data serves as the foundation of many studies. Genome sequences have been available for many Phytophthora species. Complete mitochondrial genomes from eight species have been published. These include P. infestans [P. andina, P. ipomoeae, P. mirabilis, P. phaseoli [P. nicotianae [P. sojae and P. ramorum [P. polonica, also has its complete mitochondrial genome publicly available in GenBank . It was maintained on lima bean agar. For nucleic acids extraction, it was grown in half-strength lima bean broth on a lab bench for 8 days. DNA was extracted from harvested mycelium using the Gentra Puregene Yeast/Bact kit (Qiagen).The type strain of DNA was sequenced using both PacBio and Illumina technologies. For PacBio sequencing, a 20-kb insert library was constructed and then run on two SMRT cells on the RS II sequencing platform using the P6-C4 chemistry. For Illumina sequencing, a TruSeq DNA PCR-free library with mean insert size of approximately 440 bp was constructed according to manufacturer’s instructions. It was sequenced on the HighSeq2500 platform.The PacBio reads were assembled using HGAP  and polished using Quiver . IlluminP. sansomeana was first annotated using MFannot  using the standard genetic code. Like plant mitochondria, but different from those of animal and fungi, oomycete mitochondria use the standard code [Phytophthora species were downloaded from GenBank (https://www.ncbi.nlm.nih.gov/orffinder/) was used to find open reading frames (ORFs) and BLAST searches were used to identify similarity. ORFfinder was also used to find ORFs encoding at least 100 aa in the intergenic regions of all Phytophthora mitochondrial genomes. Ribosomal RNA (rRNA) gene sequences were used to search NCBI non-redundant database and SILVA rRNA database [P. sansomeana was generated using OGDRAW [The mitochondrial genome of ard code . The aut GenBank . Proteindatabase . Transfedatabase  in the og OGDRAW . It’s gePythium ultimum [Pythium insidiosum [To construct phylogenetic tree based on the mitochondrial genomes, individual protein coding genes and rRNA genes were aligned with Muscle . The ali ultimum  and Pythsidiosum  were useP. sansomeana was assembled into a single contig of 55.5 kb using PacBio reads, with a 15.8 kb direct duplicated region at both ends, indicating a circular topology. To verify this, the contig was broken at a random position in the non-duplicated region and the two segments were re-connected by merging the duplicated regions. PacBio reads were then mapped to this re-arranged contig. Visual inspection of read alignments and coverage confirmed its circular topology. After polishing, the PacBio assembly resulted in a molecule of 39,546 bp. Illumina sequencing generated 43.3 million read-pairs at 151 bp x 2 in length. A total of 2,249,965 read-pairs were mapped to the mitochondrial genome. Based on the alignments of Illumina reads, 72 single nucleotide deletions were corrected.PacBio sequencing generated 383,775 subreads totaling 2.57 Gb after filtering and adapter removal. The N50 of subread length was 9,846 bp. The mitochondrial genome of P. sansomeana was assembled into a circular molecule of 39,618 bp with a 22.03% G+C content. It was predicted to encode 42 protein coding genes, two ribosomal RNA genes, and 25 tRNA genes. The genes were encoded by both strands. The protein coding genes included 10 NADH dehydrogenase genes in respiratory complex I , one cytochrome C reductase gene (cob) in complex III, and three cytochrome c oxidase genes in complex IV . Also included were four ATP synthase genes , five large subunit ribosomal protein genes , and 11 small subunit ribosomal protein genes  (tatC) in the twin-arginine translocation system. The annotation of this gene was inconsistent in other Phytophthora mitochondrial genomes . The six conserved ORFs with no annotated function found in other Phytophthora mitochondrial genomes , ymf98 (= orf142), ymf99 (= orf217), ymf100 (= orf100), and ymf101 (= orf64)). P. sansomeana mitochondria also included a unique ORF predicted to encode a protein of 402 aa (orf402)  . We anno genomes  were als(orf402) . No deternl) and one small subunit rRNA gene (rns). The 25 tRNA genes encoded tRNAs for 19 amino acids. There were two tRNA genes for arginine, glycine, leucine, methionine and serine, and one each for the other 14 amino acids except threonine , between nad1 and nad11 (4bp), and between tatC and ymf101 (4bp).The genome was compact. Coding regions approximately comprised 90.1% of the genome. There was no intron in any of the genes. Of the 69 intergenic regions, 53 were 50 bp or less in length and 41 were less than 20 bp. There were three overlaps: between Phytophthora species were publicly available and they were downloaded from GenBank  is a conserved ORF in all 10 Phytophthora species that encodes a protein of approximately 244 amino acids. In the 13 mitochondrial genomes of nine Phytophthora species available in GenBank  . ymf101 was not reported in P. nicotianae, but our analysis identified it at nucleotide 30206..30015.Six conserved ORFs have been reported in the mitochondrial genomes of these 10 species. Of these, 0..7628) . Ymf96 wphaseoli . InteresP. infestans haplotypes IIa and IIb, an insertion of approximately 2 kb encodes multiple ORFs [P. nicotianae, two inverted copies of orf176 in P. ramorum, and six ORFs at two location in P. sojae. Additionally, we identified orf402 in P. sansomeana and also found an ORF with 173 codons (excluding the stop codon) in P. polonica at nucleotide 540..1061 . All the genomes encoded 25 tRNA genes and these genes had the same anticodons , the section covering nad5, nad6 and trnRUCU was inverted comparing to P. ramorum (subclade 8c) and species in other clades . Species in subclade 1b was not included in this study. Within clade 1c, P. phaseoli diverged first and was basal to other species. P. andina haplotype Ia was grouped with haplotype I of P. infestans, while haplotype Ic was grouped with P. ipomoeae and P. mirabilis. These findings were in agreement with previous reports [Other than tic tree . Previou0 clades , 12. Ourar genes  and four reports , 53. TheP. sansomeana and compared it to that of nine other Phytophthora species. Inconsistencies in annotation among these mitochondrial genomes were corrected. These genomes were found to be conserved in gene content with few exceptions. Three inversion events, each covering multiple genes, were observed among these genomes. Phylogenomic analysis using the mitochondrial genomes supported or enhanced previous findings.In summary, we sequenced and annotated the complete mitochondrial genome of the soybean pathogen Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.S1 Table(XLSX)Click here for additional data file.


Brucella (B.). Proteomics has been used to investigate a few B. melitensis and B. abortus strains, but data for other species and biovars are limited. Hence, a comprehensive analysis of proteomes will significantly contribute to understanding the enigmatic biology of brucellae. For direct identification and typing of Brucella, matrix-assisted laser desorption ionization—time of flight mass spectrometry (MALDI—TOF MS) has become a reliable tool for routine diagnosis due to its ease of handling, price and sensitivity highlighting the potential of proteome-based techniques. Proteome analysis will also help to overcome the historic but still notorious Brucella obstacles of infection medicine, the lack of safe and protective vaccines and sensitive serologic diagnostic tools by identifying the most efficient protein antigens. This perspective summarizes past and recent developments in Brucella proteomics with a focus on species identification and serodiagnosis. Future applications of proteomics in these fields are discussed.Brucellosis is a global zoonosis caused by Gram-negative, facultative intracellular bacteria of the genus  Brucella (B.) encompasses 12 accepted nomo-species. The classical six species are B. abortus, B. melitensis, B. suis, B. canis, B. ovis and B. neotomae, which were primarily isolated from bovines, small ruminants, pigs, dogs, sheep and desert woodrats, respectively [B. pinnipedialis and B. ceti, were isolated from aquatic mammals [B. inopinata [B. microti [B. papionis was described as eleventh species isolated from baboons (Papio spp.) [B. vulpis was isolated from the mandibular lymph nodes of red foxes (Vulpes vulpes) [Brucellosis is one of the most frequent bacterial zoonoses spread worldwide. The infection is most often acquired via the consumption of unpasteurized milk and dairy products or direct or indirect contact with infected animals or their excretes . The mai mammals . B. inopnopinata  and B. m microti  were gaiio spp.) , and rec vulpes) .Brucella spp. are mainly associated with their survival and replication inside the host cells, including phagocytic and non-phagocytic cells. Brucellae are closely related, share noticeable similar genomes, but differ in their natural hosts, phenotypes as well as their immunogenic, proteomic and metabolomic properties [Brucella lacks classical virulence factors and several aspects of its biology e.g., pathogenesis, survival in macrophages, and host specificity, are still not well understood. To explain its virulence and host specificity, a better understanding at the proteome and metabolome level could be helpful [Brucellae are Gram-negative facultative intracellular stealthy pathogens. They can escape recognition of the innate immunity and avoid the intracellular destruction . The viroperties ,10. To doperties . Brucell helpful .B. abortus RB51 and S19, as well as B. melitensis Rev.1 [Currently, no safe human vaccine exists, while animal immunization is usually accomplished using live attenuated strains, such as is Rev.1 . Live atis Rev.1 .Brucella. Serology basically relies on the detection of anti-Brucella lipopolysaccharide (LPS) antibodies. LPS-based assays have a low specificity due to cross-reactions resulting from the similarity between immunodominant epitopes of Brucella O-polysaccharide and those of other Gram-negative bacteria resulting in a false-positive reaction. Indeed, serology based on the use of a LPS antigen does not allow the discrimination of vaccinated and infected animals. This fact raised the need to replace the LPS antigen by protein antigens. The identification of genus- or species-specific protein candidates would be useful in designing diagnostics tools, which could diagnose Brucella infection and differentiate between infections caused by different Brucella species, respectively [The diagnosis of brucellosis remains challenging and is based mainly on the serology and isolation of ectively . A comprectively .Brucella biology i.e., host-cell interaction, stress response, antibiotic targets and protein secretion [Proteomic technologies are major post-genomic approaches used to understand the consequences of regulatory processes on the protein composition of microbes. They have proven to be efficient tools to study the microbial physiology, gene expression and the interaction between the bacteria and the host cells ,18. The ecretion , as wellBrucella antibodies in milk by the milk ring test (MRT) as a field test, or by serological tools such as the slow agglutination test (SAT) and the rose bengal test (RBT), which are confirmed by the complement fixation test (CFT) and the enzyme-linked immunosorbent assay (ELISA) [Brucella to other Gram-negative bacteria. Thus, the diagnosis of Brucella based on sero-reactors should be handled with extreme caution unless accompanied by bacteriological and molecular diagnosis. Isolation, the classical biotyping and identification of Brucella spp., remains the gold standard for diagnosis, but it is hazardous, laborious, time-consuming and requires well trained personnel [Brucella at the species and biovars level requires complete bacteriological and biochemical testing or advanced molecular techniques e.g., AMOS-PCR  [Brucella species on a gel [The regular diagnosis of brucellosis relies on the indirect detection of anti- (ELISA) . One of ersonnel . The genersonnel . The chauis-PCR) , multipluis-PCR) , multipluis-PCR) , and whouis-PCR) . Both baon a gel .m/z range  and the species identification is performed by pattern-matching with the database of reference spectra of known microorganisms [m/z) of high abundant proteins and ribosomal proteins specific to the species [Brucella, taxon-specific mass spectral databases and peak pattern matching software tools are available now but additional reference spectra data on biovars are required to be created. Among several commercial software tools, the database integrated into the VITEK MS of the BioMerieuxbecame the first Brucella database validated for diagnostics with accreditation and is accessible to all users in routine diagnosis in 2010. A MALDI Biotyper (Bruker) was used to create reference spectra databases for B. melitensis, B. abortus, B. suis, B. canis, B. ceti and B. pinnipedialis [Brucella spp. including B. abortus, B. melitensis, B. suis, B. canis, B. ceti, B. inopinata, B. microti, B. ovis, B. pinnipedialis and B. neotomae [B. abortus, B. melitensis, B. suis and B. ceti were reported to be very low, while incorrect biovar assignments were found frequently except for B. suis [Brucella species with MALDI—TOF MS [In the past two decades, matrix-assisted laser desorption ionization—time of flight mass spectrometry (MALDI—TOF MS)-based typing, intact protein profiling (IPP) or intact-cell mass spectrometry (ICMS) have emerged as rapid species identification methods in the routine diagnostic laboratories ,33,34,35rganisms . The acc species . As ment species . For Bruneotomae . At the  B. suis ,32. ConsB. melitensis, B. abortus and B. suis at the species level with an accuracy of 100%, 92.9% and 100%, respectively [The difference in peak intensities due to growth conditions of the bacteria or the values of protein concentration was reported to be the least influential on species identification as the MALDI—TOF MS software tools considered only the most reproducible peaks of the generated spectra. Further improvements in terms of protein extraction protocols and the creation of unique brucellae reference databases have been reported . In a reectively .As shown in Brucella [B. abortus, B. melitensis and B. suis directly from positive blood culture flasks. The developed LC—MS/MS-based method makes use of discriminatory peptides. It considerably reduced the time required to identify the causative agents of bacteremia, and can quickly confirm the diagnoses of pathogens with a high level of accuracy.Recently, a liquid chromatography-tandem mass spectrometry (LC—MS/MS)-based method has been developed as a safe, highly accurate and unprejudiced identification method to identify highly pathogenic bacteria including Brucella . This toBrucella taxonomy [Brucella species [B. abortus and to compare those proteins with those of other vaccines and virulent strains [B. ovis and B. melitensis were investigated to identify an effective subcellular vaccine for ovine brucellosis [Brucella spp. [B. melitensis proteins. Comparative studies of protein expression for other brucellae followed [Brucella and to investigate its intracellular survival and its resistance strategy within macrophages [B. abortus, whole proteomes of virulent and of S19 vaccine strains were compared [Brucella proteomes, comparative proteomic analysis between different Brucella spp. (virulent and vaccine strains), and the examination of the differences in the protein profiles of Brucella grown under different conditions was investigated in a broad spectrum and on a large scale [Proteomics analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis (2DE), difference gel electrophoresis (DIGE) and MALDI—TOF MS remains the gold standard for protein separation and the quantification of proteins from various samples. At the end of the 20th century, SDS-PAGE profiles (protein separation based on molecular weight) and immunoblotting were used in early studies of taxonomy , and to  species ,52,53,54 strains ,49. Compcellosis . Even thcellosis . This tefollowed ,57. Subsrophages . To idencompared . At the ge scale ,46,47.Brucella proteome is limited. Differences at the proteomic level of brucellae have been investigated with different growth conditions like temperature or acidity, and oxidative or nutritional stress [B. melitensis B115 proteins was done using 2DE. The 2DE proteins map revealed 595 silver-stained protein spots and was considered the basis for comparative studies of protein expression in Brucella at that time [B. melitensis strain 16 M was done to identify and characterize the proteins expressed under laboratory conditions. Two-dimensional gel electrophoresis was used for protein separation. Of the 883 observed protein spots, 440 were identified by MALDI—TOF MS in a laboratory-grown culture. The consideration of the proteomic data with genomic sequences should lead to the identification of biochemical pathways associated with host specificity, pathogenicity, stress responses and virulence. The protein list of that study became the 2DE protein map to serve as a reference for subsequent Brucella proteomic studies [B. melitensis 16M was done to highlight the differences in protein expression with the attenuated B. melitensis Rev.1 vaccine strain. Certain metabolic pathways as well as the up and down-regulation of protein expression specific to each strain were studied, e.g., the differences in the expression of immunogenic 31-kDa outer membrane protein in Rev. 1 strain. This protein plays a role in sugar and amino acid binding, iron acquisition and lipid degradation [B. melitensis and B. abortus, were studied by 2DE and peptide mass fingerprinting via MALDI—TOF MS using laboratory-grown reference strains B. melitensis 16M and B. abortus 2308 [B. abortus infection, the proteins secreted into the cultural supernatant were studied under laboratory conditions. More than 27 soluble immunogenic proteins were detected by 2DE and mass spectrometry. Those proteins induced strong humoral and cell-mediated immune responses and could promote antibody production leading to enhanced host defense against subsequent bacterial infection [B. suis and host cell proteins connected to infection [B. abortus 2308 and its virB type IV secretion system mutant using 2DE and mass spectrometry helped to identify new T4SS-related candidate proteins [B. suis survival under extreme nutrient starvation, DIGE provided a quantitative analysis of proteomes after long-term nutrient starvation of B. suis [To date, our understanding of the l stress . In 1997hat time . The fas studies . Subsequradation ,63. Quantus 2308 . To undenfection . DIGE alnfection . This approteins . To inve B. suis . Thirty B. melitensis bacteria. Twelve differentially expressed proteins were involved in the infectious process of lung tissue [Proteomics analysis using 2DE and MALDI—TOF MS revealed that the changes in protein abundance of the infected host cells appeared between 48 and 96 h post-infection . In receg tissue . In contBrucella using gel-based tools have been reported, suffering from limitations in terms of technology and the low number of identified proteins. Recently, a comparative proteomic analysis was carried out using LC—MS based on peptide fingerprinting [B. abortus 2308 and S19 during intracellular infection was analyzed by LC—MS. The results provided insights into mechanisms utilized by brucellae to establish intracellular infection [B. abortus and B. melitensis outbreak strains from a cow and a sheep, respectively, revealed 402 differentially expressed proteins. Among them, 63 and 103 proteins were detected exclusively in the whole-cell extracts of B. abortus and B. melitensis, respectively [B. abortus was reported to have identified a total of 1221 differentially expressed proteins following multiple environmental stresses and adaptations [B. abortus 104 M human vaccine strains using high-resolution mass spectrometry revealed 1729 proteins and 218 hypothetical proteins [Brucella rough mutants ΔrfbE and ΔrfbEΔvirB123. In this study, 861 unique proteins were identified, among which 37 were differential secretory proteins [B. abortus 2308 and RB51 using liquid chromatography—tandem mass spectrometry (LC—MS/MS) succeeded to identify 220 and 171 proteins, respectively [Brucella [Proteomic analyses of various strains of printing . A signinfection . The labectively . The masptations . Furtherproteins . The liqproteins . The proectively . LC—MS rBrucella species [Brucella immunoproteomics have focused on the use of reference strains and experimentally produced hyper-immune sera [Brucella sera revealed different spectra of immunoreactive proteins. When a protein sample of B. melitensis B115 was tested using monoclonal antibodies (MAbs), 25 protein spots out of 595 spots separated on a 2DE plot were stained [B. ovis with a serum of a sheep naturally infected with B. ovis. The combination of 2DE and protein microsequencing succeeded to identify 82 immunoreactive cytoplasmic, periplasmic and membrane protein spots [B. abortus 1119-3 whole-cell lysate was tested with rabbit hyperimmune sera, 17 out of 383 protein spots were stained. Using the surface-enhanced laser desorption/ionization mass spectrometry (SELDI-MS), these immunogenic spots were assigned to six proteins: Cu-Zn SOD, BCSP31, GroEL, GroES, L7/L12 and DnaK [B. abortus 2308 cell envelope (CE) using antiserum from bovine and a human patient infected with B. suis was carried out. Among the 163 identified proteins, several new immunogenic proteins such as fumarate reductase flavoprotein subunit, cysteine synthase A and F0F1-type ATP synthase α-subunit were found. The authors assumed that those proteins were suitable candidates for developing vaccines against brucellosis in humans and bovines [Brucella sera. A lysate of the B. melitensis M5 vaccine strain was tested against pooled bovine sera, and 88 immunoreactive protein spots were detected and assigned to 61 proteins by MALDI—TOF MS, including elongation factor G, F0F1 ATP synthase ß-subunit beta and OMP1. Only eight were virulence-related proteins [B. melitensis 16M cell lysate tested against pooled human and goat antiserum produced 23, 33 and 11 immunoreactive protein spots with sera of humans, goats or both, respectively. Two interesting riboflavin synthase α chain (RS-α) and Loraine synthase (LS-2) proteins were detected using antisera obtained from Brucella-infected humans and goats. Both proteins (rRS-α and rLS-2-) provided partial protection in mice challenged with B. melitensis [B. abortus 2308 revealed 18 immunogenic insoluble proteins. The spots were reactive against anti-Brucella antisera but not towards negative sera and anti Y. enterocolitica sera [Brucella spp. and stage of infection. Routine serodiagnosis fails to detect the stage of infection, but proteomic analysis shows time-course-dependent B. abortus protein patterns when a subsequent set of sera is used [Despite the high genomic similarity among  species , it has  species ,77,78. Tune sera ,81,82,83une sera ,78. As s stained . The proin spots . When thand DnaK . To iden bovines . Thirty- is used .B. abortus 544 were found to be reactive at day 10, 30 and 60 post-infection when tested with sera from experimentally infected mice in 2DE immunoblotting, respectively [B. abortus 544 protein lysates have been tested with sera from B. abortus-infected cattle at week 3, week 7 and week 10 post-infection. Thus, 134, 110 and 106 proteins were reactive, respectively, and 55 antigens were predominant at all three-time points [Brucella biovars and different sets of sera is essential to learn about the brucellae proteome and host immune responses to promote the development of advanced diagnostics and novel vaccine candidates [Prominent immunogenic proteins show time course-dependent reactions. It was found that 13, 24 and 55 proteins of ectively . In the e points . Therefondidates ,84.B. abortus and B. melitensis field strains against field sera collected from cattle, buffaloes, sheep and goats was performed with SDS-PAGE and Western blotting. The results showed that Brucella appeared to express heat shock proteins, enzymes, binding proteins and hypothetical proteins for their survival in the host during the early stage of infection [B. abortus and 20 proteins of B. melitensis were distinctly immunoreactive. Some were host-specific and others crossed the host species barrier. Three proteins from B. abortus , and additionally, the amino acid ABC transporter substrate-binding protein from B. melitensis and fumarylacetoacetate hydrolase from both Brucella spp. were immunoreactive with the sera of naturally infected cattle, buffaloes, sheep and goats. The identified proteins were supposed to be used for the design of serological assays able to detect pan-Brucella, B. abortus- and B. melitensis-specific antibodies [Immunoblotting of whole-cell protein extracts of nfection . When 2Dtibodies . In addiB. abortus have been proposed to be potential candidates for subunit vaccines [Recently, several studies have been carried out to evaluate immunogenicity and protective responses, as well as the application of immunogenic proteins in the diagnosis of brucellosis ,85. For vaccines . Howevervaccines . HoweverBrucella research. The earliest works begun with the protein separation by SDS-PAGE and 2DE, i.e., the manual and mechanical excision of protein spots and the identification of proteins using MALDI—TOF MS. In recent years, LC- MS-based proteomics technology has been developed and is widely applied in Brucella research. It is capable of identifying and quantifying higher numbers of proteins. Immunoproteomics utilizing 2DE—Western blotting is one of the most common tools implemented to identify antigens for serodiagnostics and the development of recombinant vaccine candidates. In the last decade, the development of LPS-free and protein-based antigens for serodiagnostics or vaccine design has attracted researchers worldwide and became a central target in the Brucella research. Several cell surface proteins and intracellular components of different Brucella spp. have been identified and classified as protective antigens. However, they most often provide only weak immunogenic responses. Several databases have been developed to be used for the direct identification and classification of brucellae. Now the technology is used in routine microbiological work and is the first line diagnosis. The use of proteomics as a comprehensive biological approach has contributed to rapid species identification and a better understanding of various aspects of the life cycle of brucellae. However, the current knowledge on proteomes of Brucella is mostly based on the examination of type strains or a limited number of isolates from a single host. This lack of data results in a somewhat sketchy picture of the whole proteome of Brucella and needs to be closed by pan-proteomics studies. Thus, the future of Brucella proteomics should focus on whole and pan proteomes [Brucella like host specificity, the interaction with the host immune system, the development of resistance or intracellular survival are enigmatic. The use of proteomic approaches has already contributed to unravel and elucidate some aspects of the pathogenesis of Brucella. Proteome studies involving isolates of virulent and avirulent Brucella species and biovars in combination with multitudes of infection models will enable us to develop better diagnostics and safer vaccines in the future to combat brucellosis in humans and animals successfully.Proteomic technologies are major post-genomic approaches used to understand the consequences of regulatory processes on the protein composition of microbes. It has proven as a handy tool to study the microbial physiology, gene expression and the interaction between bacteria and their host cells. Since the beginning of the 21st century, proteomics technology has been applied to unravel various enigmatic areas of roteomes . Compara


Asarum sieboldii is a medicinal plant belonging to the Aristolochiaceae family. In this study, complete chloroplast genome sequence of A. sieboldii was characterized through de novo assembly with next generation sequencing data. The chloroplast genome is 193,356 bp long and has the stereotypical tripartite organization consisting of large single copy region and a pair of inverted repeats. The genome contains 78 protein-coding genes, 30 rRNA genes, and 4 tRNA genes. Phylogenetic analysis revealed that A. sieboldii has close relationship with Piper coenoclatum . Asarum L. (Aristolochiaceae), which consists of approximately 100 species, is usually distributed in temperate regions of Northern Hemisphere, with the center of diversity in Eastern Asia  of Korean Asarum species have been sequenced and analyzed for species identification and discrimination in genus Asarum  The genus  Asia Oh . Since aina Zhou . AlthougA. sieboldii to contribute to the classification and development of DNA markers for authentication of Asarum species. The specimen was collected from Mt Deogyu, South Korea  and deposited at NIBR herbarium (KB) with the accession number NIBR-VP0000640510. Genome was sequenced using the Illumina MiSeq platform  and high-quality paired-end reads of ca. 1.7 Gb were used to assemble chloroplast genome (GenBank Accession no. MG551543), as described previously  of a 48,401bp IR separating an LSC region of 96,554bp. The SSC region of A. sieboldii is reverse-tandemly duplicated and then integrated into two IRs . The A. sieboldii genome contains 78 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. The total number of identified genes encoded is 112 genes, with 12 genes duplicated within the SSC integrated into IRs .The chloroplast genome was 193,356 bp in length with 36.2% overall GC content. Except the chloroplast genome of A. sieboldii with other taxa, a neighbor-joining (NJ) tree was constructed using 62 common protein-coding genes of A. sieboldii and 25 vascular plant taxa .To understand phylogenetic relationship of ant taxa . The NJ es order . ChloropClick here for additional data file.


Eupatorium adenophorum have widespread invaded the karstecosystem of southwest China and threatened the regional native communitystability. Arbuscular mycorrhizae (AM) plays an important role in promotinggrowth for host plants via root external mycelia. However, whether AM regulatesplant root traits underlying competition between invasive and native species viamycorrhizal networks in karst habitats, remains unclear. An experiment wasconducted in a microcosm composed of two planting compartments flanking acompetition compartment. The invasive E.adenophorum and native Artemisia annuawere each placed in one of the two planting compartments with or withoutGlomus etunicatum fungus. The nutrient access treatmentsincluded the competitive utilization (Cu), single utilization (Su) andnon-utilization (Nu) by using different nylon meshes allowed or preventedmycelium passing to acquire nutrients from the competition compartment. Roottraits and nutrients of the two species were analyzed. The results showed thatAM fungi had differential effects on root traits and nutrients ofE. adenophorum and A.annua seedlings, which increased dry weight, length,surface area, volume, tips and branching points in roots, specific root lengthand volume, root nitrogen (N) and phosphorus (P) contents under Cu, Su and Nutreatments. AM fungus was also associated with decreases in the average diameterfor both species. Under the Cu treatment, E.adenophorum had significantly greater length, surface area,volume, tips and branching points of roots, specific root traits, and root N andP than A. annua. AM fungi changed rootphenotypes and nutrient uptake for both invasive and native plant species viainterconnected mycorrhizal networks. Overall, our results suggest that throughmycorrhizal networks, the invasive plant experiences greater benefits than thenative plant in the nutrient competition, which fosters root morphologicaldevelopments in karst soil.The  Eupatoriumadenophorum have successfully invaded the karst habitat in southwestChina and have spread continuously [Karst landforms develop from carbonate rock and are widely distributed in southwestChina . This hainuously , seriousinuously . Harner inuously  argued tinuously , 8 also E.adenophorum and can enhance the competitive effect ofE. adenophorum on native plants [Solidago canadensis and native Kummerowastriata are interconnected with an arbuscular mycorrhizal network. Whenresources are scarce, there will be competition for aboveground light andcompetition for belowground water and mineral nutrients among plant species [Arbuscular mycorrhizal (AM) fungi are functional microorganisms that can formmutually beneficial symbiotic relationships with more than 80% of land plants . An arbue plants . Awaydule plants  discover species , 16. How species .The nut species , 19.Mea species . Thus th species .Roots can enhance a plant’s competitive advantage by adjusting its morphology whentwo adjacent plants compete for limited soil resources . AdditioA microcosm experiment was set up using devices with three compartments  in the g+) includedinoculation with 50g Glomus etunicatum inoculum , containing probable500 spores as well as hyphae and colonized root pieces. The nutrient accesstreatments for the competition compartment interconnecting two plantingcompartments by mycorrhizal networks, which used 20um nylon mesh and 0.45umnylon mesh in different ways. As follows: (1) the competitive utilizationnutrient treatment (Cu) was to use the 20μm double-nylon mesh on the baffleplate of the central competition compartment and the planting compartments onboth sides, allowing the AM mycelia in planting compartments ofE. adenophorum and A.annua commonly entering into the competition compartment,and form an interconnected mycorrhizal networks among all three compartments;(2) the single utilization nutrient treatment (Su) used 20μm double-nylon meshon one side of the baffle plate between the competition compartment and oneplanting compartment and a 0.45μm nylon mesh on the other side between thecompetition compartment and the other planting compartment, allowing the AMmycelium of one planting compartment of E.adenophorum or A. annuato pass through the competition compartment to utilize nutrients. (3) thenon-utilization of nutrients treatment (Nu) which in contrast to Cc and Su, usedthe 0.45μm nylon mesh to separate both planting compartments from the centralcompartment, so that the nutrient resources of the competition compartment couldnot be utilized by both species as their associated mycelia could not cross thefine 0.45μm nylon mesh.This experiment had both mycorrhizal fungus treatments and nutrient accesstreatments. The mycorrhizal fungus treatments  [E. adenophorum and A.annua were collected from Guanling county of Guizhouprovince of China, a typical desertification area within a karst ecosystem. Inour previous field survey, the E. adenophorumhas severely invaded the karst area of southwest China and coexists with nativeA. annua, and both species are herbaceousplants of Asteraceae family and have a similar niche [2O2) solutionfor 10 minutes and repeatedly washed with sterile water three times. Eachcompartment was filled with 2.5 kg of the sterilized substrate, and five seedsof plants were taken into planting compartments, respectively. 50g of inoculumwas added into the planting compartments as M+ treatments. Theinoculum had been propagated with Trifolium repens for fourmonths, which was sterilized at 0.14 Mpa, at 126 °C for one hour beforeinoculation with Glomus etunicatum. Additionally, M-treatments received an additional 10 ml of the filtrate by weighing 50g ofGlomus etunicatum inoculum and by filtering it withultrapure water, and double-layer filter paper, along with a 50g of autoclavedinoculum was added, in order to ensure the same microflora in M- andM+ except for the target fungus (Glomusetunicatum). After ten days of seedling growth, two seedlings werekept in each planting compartment. Each treatment used six replicates. Allexperimental materials were cultured in a greenhouse for 12 weeks and then wereharvested for measurement.The plant growing substrate was a mixture of limestone soil and sand by volumeratio of 3:1, which had pH 7.45, total nitrogen 2.27 g. kgn (2005) . The subar niche . The seeE. adenophorum andA. annua was determined by weighing rootmaterial after drying at 80°C for constant weight. Plant N was determined by theKjeldahl method and P was determined by Molybdenum-Antimony colorimetry [The determination of mycorrhizal colonization rate adopted methods described byHe and Zhong (2012) .The rooorimetry . Plant rorimetry . The roo+ andM- or treatments of Cu, Su and Nu treatments in length, averagediameter, surface area, volume, tips and branching points of roots, specificroot length, area and volume, and root N and P contents per length, area andvolume by the least significant difference (LSD). Three-way ANOVAs were appliedfor the effects of species origin (E.adenophorum (invasive) vs. A.annua (native)), mycorrhizal fungus (M+ vs.M-) and nutrient access treatments (Cu vs. Su vs. Nu) and theirinteractions on the root traits of morphology and nutrients. Origin 8.0 softwarewas used to the bar graphs.Statistical analyses were performed using the SPSS 18.0 software. All of the datawere tested for normality and homogeneity of variance before analysis. Varianceanalysis was applied to compare differences between M+ treatment, the mycorrhizal colonization rates ofE. adenophorum and A.annua were not significantly different among Cu, Su and Nutreatments, while no AM colonization was observed under the M–. Themycorrhizal colonization rate of E.adenophorum was significantly greater thanA. annua under the Cu, Su, and Nutreatments, indicating that E. adenophorum ismore responsive to mycorrhizal colonization than A.annua ,and in average diameter under Cu and Nu (M+ < M-), the average diameter of the Cu treatment was significantlylower than in Nu, and the surface area of the Su and Cu treatments wassignificantly higher than in Nu; under M-, there were no significantdifferences in all these root morphological indexes among Cu, Su, and Nutreatments  and AM fungus (M) significantly affected the morphological traitsof roots . ForE. M+ < M-). ThenutM+ < M-). ForE. eatments . ForA. an in Nu . Theintan in Nu .+, the E.adenophorum was significantly greater than theA. annua in length under the Cu and Su,and in surface area and volume under the Cu, Su and Nu treatments; but theaverage diameter of E. adenophorum was lowerthan A. annua under the Cu and Su treatments;under M-, the volume of E.adenophorum was lower than A.annua under the Cu treatment  in numbers ofroot tips and branching points of E.adenophorum and A. annuaunder the Cu, Su and Nu treatments  and AM fungus (M) significantly affected the numbers of root tipsand branching points of plants . A signieatments . The nuteatments . Under Meatments . Interes with M- . Overallr the Cu . This reE. adenophorum, a significant differencewas observed between M+ and M- (M+ >M-) in specific root length under the Cu and Su treatments, inspecific root area under the Cu, and in specific root volume under the Cu, Suand Nu treatments; for A. annua, there was asignificant difference between M+ and M- (M+> M-) in specific root length under the Cu, Su and Nu treatmentsand in specific root volume under the Su , specific root volume in Cu and Su was significantly greater than inthe Nu under M+; for A. annua, allspecific root traits of the Su treatment was significantly higher than in Cu andNu under M+  and AM fungus (M) significantly affected the specific root traitsof plants . ForE. r the Su . The nutr the Su . ForE. under M+ . The intt volume . Generaleatments. TheserE. adenophorum, a significant differencewas observed between M+ and M- (M+ >M-) in root N content per length under the Su treatment, in rootN content per area and volume under the Cu and Su, and in root P content perlength, area and volume under the Cu, Su and Nu treatments  in root Ncontent per area under the Su and Nu treatments, in root N content per volumeunder the Su treatment, and in root P per area and volume under the Cu, Su andNu treatments  and AM fungus (M) significantly affected N and P contents inroots of plants .For E. eatments . For A.eatments .The nuteatments . Under Mnthe Su . Besides with M- . General N and P . It indiE.adenophorum and native A.annua in this experiment  [Cyclobalanopsis glauca in karst rocky desertification areas.Root morphology can reveal a plant’s ability to absorb nutrients [+ compared with M-. This is similar to Li et al. (2016)[AM fungi differentially increased the dry weight, length, surface area, volume, tips,branching points and N and P contents in roots for invasive ent Figs . Previouvimineum ; Huang evimineum  also dis. (2015)  showed tutrients , and difutrients , 51. Ourutrients –54. Inte. (2016) suggest . (2016) also docE.adenophorum and A. annuaseedlings  revealedngs Figs  and 4a, nophorum  and furtnditions , which irst soil .E.adenophorum and native A.annua in the Su treatment were greater than the Nu treatment(Figs E. adenophorum in order to obtaininga greater competitive advantage for that species than A.annua under the Cu treatment in karst soil (Figs AM fungi affected plant competition on nutrient uptake  and enhaentFigs  and 6, woil Figs  and 6. TE. adenophorum and A.annua in Su treatment were larger than in the Nu treatment(Figs Cinnamomum camphora root phenotypes in karst areas; and theseroot phenotypic indices of E. adenophorum andA. annua from Figs E.adenophorum were significantly higher than A.annua under the Cu treatment (Figs E. adenophorum may have greater rootmorphological plasticity response over A. annua toenhance its competitiveness in order to absorb and utilize the soil resources inkarst habitats. Root diameter size determines the utilization efficiency of plantroots for belowground resources, and the uptake capacity of nutrients and water bythe smaller diameter roots is higher than the thicker diameter roots [E.adenophorum was significantly lower than A.annua, and the specific root length and area was significantlygreater than A. annua under the Cu condition (FigsE.adenophorum in nutrient and water acquisition over nativeA. annua root in the fragile karst ecosystemsof southwest China.Additionally, AM fungi will inevitably cause changes in plant phenotype whileimproving plant nutrients . In thisentFigs –5. Theseent Figs , which ier roots . Meanwhier roots . In our ion Figs, suggestE. adenophorum and nativeA. annua; and the root average diameter ofboth species was decreased. Many of the observed increases were more significant forE. adenophorum than for A.annua. In conclusion, based on these findings, we suggest thatthe invasive plant experienced greater benefits than the native plant in nutrientacquisition and for root traits and root system developments when in associationwith an AM mycorrhizal network in karst habitats.In this experiment, AM fungus was associated with changes in root traits andincreased nutrient uptake for invasive and native plant species via theinterconnective mycorrhizal networks. Specifically, a large number of root traitswere enhanced including root dry weight, length, surface area, volume, number oftips and branching points, specific root length and volume, root N and P contentsfor invasive