Data_Sheet_1_Isolation and identification of antagonistic Bacillus amyloliquefaciens HSE-12 and its effects on peanut growth and rhizosphere microbial community.docx

The HSE-12 strain isolated from peanut rhizosphere soil was identified as Bacillus amyloliquefaciens by observation of phenotypic characteristics, physiological and biochemical tests, 16S rDNA and gyrB gene sequencing. In vitro experiments showed that the strain possessed biocontrol activity against a variety of pathogens including Sclerotium rolfsii. The strain has the ability to produce hydrolytic enzymes, as well as volatile organic compounds with antagonistic and probiotic effects such as ethyleneglycol and 2,3-butanediol. In addition, HSE-12 showed potassium solubilizing (10.54 ± 0.19 mg/L), phosphorus solubilization (168.34 ± 8.06 mg/L) and nitrogen fixation (17.35 ± 2.34 mg/g) abilities, and was able to secrete siderophores [(Ar-A)/Ar × 100%: 56%] which promoted plant growth. After inoculating peanut with HSE-12, the available phosphorus content in rhizosphere soil increased by 27%, urease activity increased by 43%, catalase activity increased by 70% and sucrase activity increased by 50% (p < 0.05). The dry weight, fresh weight and the height of the first pair of lateral branches of peanuts increased by 24.7, 41.9, and 36.4%, respectively, compared with uninoculated peanuts. In addition, compared with the blank control, it increased the diversity and richness of peanut rhizosphere bacteria and changed the community structure of bacteria and fungi. The relative abundance of beneficial microorganisms such as Sphingomonas, Arthrobacter, RB41, and Micromonospora in rhizosphere soil was increased, while the relative abundance of pathogenic microorganisms such as Aspergillus, Neocosmospora, and Rhizoctonia was decreased.</p

Image_1_Isolation and identification of antagonistic Bacillus amyloliquefaciens HSE-12 and its effects on peanut growth and rhizosphere microbial community.JPEG

The HSE-12 strain isolated from peanut rhizosphere soil was identified as Bacillus amyloliquefaciens by observation of phenotypic characteristics, physiological and biochemical tests, 16S rDNA and gyrB gene sequencing. In vitro experiments showed that the strain possessed biocontrol activity against a variety of pathogens including Sclerotium rolfsii. The strain has the ability to produce hydrolytic enzymes, as well as volatile organic compounds with antagonistic and probiotic effects such as ethyleneglycol and 2,3-butanediol. In addition, HSE-12 showed potassium solubilizing (10.54 ± 0.19 mg/L), phosphorus solubilization (168.34 ± 8.06 mg/L) and nitrogen fixation (17.35 ± 2.34 mg/g) abilities, and was able to secrete siderophores [(Ar-A)/Ar × 100%: 56%] which promoted plant growth. After inoculating peanut with HSE-12, the available phosphorus content in rhizosphere soil increased by 27%, urease activity increased by 43%, catalase activity increased by 70% and sucrase activity increased by 50% (p < 0.05). The dry weight, fresh weight and the height of the first pair of lateral branches of peanuts increased by 24.7, 41.9, and 36.4%, respectively, compared with uninoculated peanuts. In addition, compared with the blank control, it increased the diversity and richness of peanut rhizosphere bacteria and changed the community structure of bacteria and fungi. The relative abundance of beneficial microorganisms such as Sphingomonas, Arthrobacter, RB41, and Micromonospora in rhizosphere soil was increased, while the relative abundance of pathogenic microorganisms such as Aspergillus, Neocosmospora, and Rhizoctonia was decreased.</p

Methodic of basic punches and defences in box.

Title: Methodic ofbasic punches and defences in box. The aim of diploma theses: Create a manual ofmethodic ofbasic punches and defences in box. Method: In making my theses I used usual methods like dokument analysis, interrogation and observation. I replenished this acquired information with study of Czech and foreign literature, reading magazines and searching on internet. Also I completed this infonnation with my own experience. Results: In this diploma theses I described all of basic punches and defences in box. Process is systematic from basic pose and simply punches till offensive combinations, active and passive defence. Keywords: Box, punch, defence, offensive.

Wild-type and transformants of <i>Fusarium graminearum</i> strains used in this study.

<p>Wild-type and transformants of <i>Fusarium graminearum</i> strains used in this study.</p

The S289 mutation affected the function but not localization of FgPrp4.

<p>(<b>A).</b> Western blots of total proteins isolated from the wild type (PH-1) and the <i>FgPRP4</i>-3xFLAG transformant were detected with an anti-3xFLAG antibody. (<b>B).</b> 12 h germlings of the <i>Fgprp4/FgPRP4</i>^S289A transformant FPA2 were examined by DIC and epifluorescence microscopy. Bar = 20 μm. <b>(C).</b> Three-day old PDA cultures of the <i>Fgprp4</i> mutant (FP1), complemented transformant (FPN1), and <i>Fgprp4/FgPRP4</i>^S289A transformant (FPA2).</p

Effects of <i>FgPRP4</i> deletion on intron splicing.

<p>(<b>A).</b> Box-plot comparison of intron retention levels between the wild type (PH-1) and <i>Fgprp4</i> mutant (FP1) in replica experiments. The statistical significance for each comparison is analyzed by <i>t</i>-test (****, <i>P</i><0.0001). (<b>B).</b> The percentage of introns and genes with the three marked intron retention levels in <i>Fgprp4</i> compared to the wild type. (<b>C)</b>. Introns that were increased in intron retention over 2-fold in <i>Fgprp4</i> in both replica experiments. (<b>D).</b> Intron splicing defects in the labelled genes were verified by RT-PCR with primers flanking the introns with reduced splicing efficiency (marked with *) in the <i>Fgprp4</i> mutant. Lanes 1–3 were PCR results with the genomic DNA, cDNA of PH-1, and cDNA of <i>Fgprp4</i>, respectively. The sizes of amplified bands are labelled on the side.</p

Assays for the function of the N-terminal 310 aa of FgPrp4.

<p>(<b>A).</b> Conidia, 12 h germlings, and hyphae of the <i>Fgprp4/FgPRP4-</i>GFP transformant (FPN1) were examined by DIC and epifluorescence microscopy. Bar = 20 μm. (<b>B).</b> The expression level of <i>FgPRP4</i> was assayed by qRT-PCR with RNA isolated from conidia, 12 h germlings, perithecia at 10 days post-fertilization, and infected wheat heads at 7 days post-inoculation (dpi). Mean and standard deviation were calculated with data from three independent biological replicates. The β-tubulin gene FGSG_06611 of <i>F</i>. <i>graminearum</i> was used as the internal control. (<b>C).</b> Three-day old PDA cultures of the <i>Fgprp4</i> mutant (FP1), <i>Fgprp4/FgPRP4</i>-GFP transformant (FPN1), and <i>Fgprp4/FgPRP4</i>^Δ1-310-GFP transformant (FPN310). (<b>D).</b> 12 h germlings of transformant FPN310 were examined by DIC and epifluorescence microscopy. Bar = 20 μm.</p

Schematic draw of the pre-mRNA splicing processes and components of tri-snRNP related to this study.

<p>Exons and one intron are represented by boxes and solid line, respectively. Base pairing of U1 to 5’ss and recognition of BP by U2 (formation of complex A) are followed by the integration of preformed U4/U6-U5 tri-snRNP to form complex B. Whereas Prp4 and Prp31 are components of U4/U6 snRNP, Brr2, Prp8, and Prp6 are components of U5 snRNP. Phosphorylation of Prp6 and Prp31 by Prp4 is associated with the activation of B-complex (complex B^act). U1, U4, Prp4, Prp6, and Prp31 are released from the activated spliceosome that catalyzes two sequential transesterifications reactions for intron splicing.</p

Two Cdc2 Kinase Genes with Distinct Functions in Vegetative and Infectious Hyphae in <i>Fusarium graminearum</i>

<div><p>Eukaryotic cell cycle involves a number of protein kinases important for the onset and progression through mitosis, most of which are well characterized in the budding and fission yeasts and conserved in other fungi. However, unlike the model yeast and filamentous fungi that have a single Cdc2 essential for cell cycle progression, the wheat scab fungus <i>Fusarium graminearum</i> contains two <i>CDC2</i> orthologs. The <i>cdc2A</i> and <i>cdc2B</i> mutants had no obvious defects in growth rate and conidiation but deletion of both of them is lethal, indicating that these two <i>CDC2</i> orthologs have redundant functions during vegetative growth and asexual reproduction. However, whereas the <i>cdc2B</i> mutant was normal, the <i>cdc2A</i> mutant was significantly reduced in virulence and rarely produced ascospores. Although deletion of <i>CDC2A</i> had no obvious effect on the formation of penetration branches or hyphopodia, the <i>cdc2A</i> mutant was limited in the differentiation and growth of infectious growth in wheat tissues. Therefore, <i>CDC2A</i> plays stage-specific roles in cell cycle regulation during infectious growth and sexual reproduction. Both <i>CDC2A</i> and <i>CDC2B</i> are constitutively expressed but only <i>CDC2A</i> was up-regulated during plant infection and ascosporogenesis. Localization of Cdc2A- GFP to the nucleus but not Cdc2B-GFP was observed in vegetative hyphae, ascospores, and infectious hyphae. Complementation assays with chimeric fusion constructs showed that both the N- and C-terminal regions of Cdc2A are important for its functions in pathogenesis and ascosporogenesis but only the N-terminal region is important for its subcellular localization. Among the Sordariomycetes, only three Fusarium species closely related to <i>F</i>. <i>graminearum</i> have two <i>CDC2</i> genes. Furthermore, <i>F</i>. <i>graminearum</i> uniquely has two Aurora kinase genes and one additional putative cyclin gene, and its orthologs of <i>CAK1</i> and other four essential mitotic kinases in the budding yeast are dispensable for viability. Overall, our data indicate that cell cycle regulation is different between vegetative and infectious hyphae in <i>F</i>. <i>graminearum</i> and Cdc2A, possibly by interacting with a stage-specific cyclin, plays a more important role than Cdc2B during ascosporogenesis and plant infection.</p></div

Candidate Prp4-target genes sequenced in the selected suppressor strains.

<p>Candidate Prp4-target genes sequenced in the selected suppressor strains.</p