Project description:The establishment of the harmful pathogen Fusarium graminearum in different agroecosystems may strongly depend on the ability of the soils to suppress its development and survival. This study aimed to evaluate the influence of different soil tillage systems (i.e., conventional tillage, reduced tillage and no-tillage) on soil fungistasis against F. graminearum. Soil samples were collected three times during the plant growing season in 2016 and 2017 from a long-term, 20-year soil tillage experiment. The F. graminearum in the soil samples was quantified by real-time qPCR. The soil fungistasis was evaluated by the reduction in the radial growth of F. graminearum in an in vitro assay. The antagonistic activity of the soil bacteria was tested using the dual culture method. The F. graminearum DNA contents in the soils were negatively correlated with soil fungistasis (r = -0.649 *). F. graminearum growth on the unfumigated soil was reduced by 70-87% compared to the chloroform fumigated soil. After the plant vegetation renewal, the soil fungistasis intensity was higher in the conventionally tilled fields than in the no-tillage. However, no significant differences were obtained among the tillage treatments at the mid-plant growth stage and after harvesting. 23 out of 104 bacteria isolated from the soil had a moderate effect, and only 1 had a strong inhibitory effect on the growth of F. graminearum. This bacterium was assigned 100% similarity to the Bacillus amyloliquefaciens Hy7 strain (gene bank no: JN382250) according to the sequence of the 16S ribosome subunit coding gene. The results of our study suggest that the presence of F. graminearum in soil is suppressed by soil fungistasis; however, the role of tillage is influenced by other factors, such as soil biological activity, type and quantity of plant residues and environmental conditions.
Project description:The filamentous fungus Fusarium graminearum, a devastating pathogen of barley (Hordeum vulgare L.), produces mycotoxins that pose a health hazard. To investigate the surface interactions of F. graminearum on barley, we focused on barley florets, as the most important infection site leading to grain contamination. The fungus interacted with silica-accumulating cells (trichomes and silica/cork cell pairs) on the host surface. We identified variation in trichome-type cells between two-row and six-row barley, and in the role of specific epidermal cells in the ingress of F. graminearum into barley florets. Prickle-type trichomes functioned to trap conidia and were sites of fungal penetration. Infections of more mature florets supported the spread of hyphae into the vascular bundles, whereas younger florets did not show this spread. These differences related directly to the timing and location of increases in silica content during maturation. Focal accumulation of cellulose in infected paleae of two-row and six-row barley indicated that the response is in part linked to trichome type. Overall, silica-accumulating epidermal cells had an expanded role in barley, serving to trap conidia, provide sites for fungal ingress and initiate resistance responses, suggesting a role for silica in pathogen establishment.
Project description:We constructed a genetic linkage map of Gibberella zeae (Fusarium graminearum) by crossing complementary nitrate-nonutilizing (nit) mutants of G. zeae strains R-5470 (from Japan) and Z-3639 (from Kansas). We selected 99 nitrate-utilizing (recombinant) progeny and analyzed them for amplified fragment length polymorphisms (AFLPs). We used 34 pairs of two-base selective AFLP primers and identified 1048 polymorphic markers that mapped to 468 unique loci on nine linkage groups. The total map length is approximately 1300 cM with an average interval of 2.8 map units between loci. Three of the nine linkage groups contain regions in which there are high levels of segregation distortion. Selection for the nitrate-utilizing recombinant progeny can explain two of the three skewed regions. Two linkage groups have recombination patterns that are consistent with the presence of intercalary inversions. Loci governing trichothecene toxin amount and type (deoxynivalenol or nivalenol) map on linkage groups IV and I, respectively. The locus governing the type of trichothecene produced (nivalenol or deoxynivalenol) cosegregated with the TRI5 gene (which encodes trichodiene synthase) and probably maps in the trichothecene gene cluster. This linkage map will be useful in population genetic studies, in map-based cloning, for QTL (quantitative trait loci) analysis, for ordering genomic libraries, and for genomic comparisons of related species.
Project description:Fungal viruses (mycoviruses) have attracted more attention for their possible hypovirulence (attenuation of fungal virulence) trait, which may be developed as a biocontrol agent of plant pathogenic fungi. However, most discovered mycoviruses are asymptomatic in their hosts. In most cases, mycovirus hypovirulent factors have not been explored clearly. In this study, we characterized a ssRNA mycovirus in Fusarium graminearum strain HB56-9. The complete nucleotide genome was obtained by combining random sequencing and rapid amplification of cDNA ends (RACE). The full genome was 6621-nucleotides long, excluding the poly(A) tail. The mycovirus was quite interesting because it shared 95.91% nucleotide identities with previously reported Fusarium graminearum virus 1 strain DK21 (FgV1-DK21), while the colony morphology of their fungal hosts on PDA plates were very different. The novel virus was named Fusarium graminearum virus 1 Chinese isolate (FgV1-ch). Like FgV1-DK21, FgV1-ch also contains four putative open reading frames (ORFs), including one long and three short ORFs. A phylogenetic analysis indicated that FgV1-ch is clustered into a proposed family Fusariviridae. FgV1-ch, unlike FgV1-DK21, had mild or no effects on host mycelial growth, spore production and virulence. The nucleotide differences between FgV1-ch and FgV1-DK21 will help to elucidate the hypovirulence determinants during mycovirus-host interaction.
Project description:In nature, fungal endophytes often have facultative endohyphal bacteria (FEB). Can a model plant pathogenic fungus have them, and does it affect their phenotype? We constructed a growth system/microcosm to allow an F. graminearum isolate to grow through natural soil and then re-isolated it on a gentamicin-containing medium, allowing endohyphal growth of bacteria while killing other bacteria. F. graminearum PH-1 labelled with a His1mCherry gene staining the fungal nuclei fluorescent red was used to confirm the re-isolation of the fungus. Most new re-isolates contained about 10 16SrRNA genes per fungal mCherry gene determined by qPCR. The F. graminearum + FEB holobiont isolates containing the bacteria were sub-cultured several times, and their bacterial contents were stable. Sequencing the bacterial 16SrRNA gene from several Fg-FEB holobiont isolates revealed endophytic bacteria known to be capable of nitrogen fixation. We tested the pathogenicity of one common Fg-FEB holobiont association, F. graminearum + Stenatrophomonas maltophilia, and found increased pathogenicity. The 16SrRNA gene load per fungal His1mCherry gene inside the wheat stayed the same as previously found in vitro. Finally, strong evidence was found for Fg-S. maltophilia symbiotic nitrogen fixation benefitting the fungus.
Project description:Fusarium graminearum virus 2 (FgV2) infection induces phenotypic changes like reduction of growth rate and virulence with an alteration of the transcriptome, including various transcription factor (TFs) gene transcripts in Fusarium graminearum. Transcription factors are the primary regulator in many cellular processes and are significant in virus-host interactions. However, a detailed study about specific TFs to understand interactions between FgV2 and F. graminearum has yet to be conducted. We transferred FgV2 to a F. graminearum TF gene deletion mutant library to identify host TFs related to FgV2 infection. FgV2-infected TF mutants were classified into three groups depending on colony growth. The FgV2 accumulation level was generally higher in TF mutants showing more reduced growth. Among these FgV2-infected TF mutants, we found several possible TFs that might be involved in FgV2 accumulation, generation of defective interfering RNAs, and transcriptional regulation of FgDICER-2 and FgAGO-1 in response to virus infection. We also investigated the relation between FgV2 accumulation and production of reactive oxygen species (ROS) and DNA damage in fungal host cells by using DNA damage- or ROS-responsive TF deletion mutants. Our studies provide insights into the host factors related to FgV2 infection and bases for further investigation to understand interactions between FgV2 and F. graminearum.
Project description:BackgroundFusarium graminearum virus 1 strain-DK21 (FgV1-DK21) is a mycovirus that confers hypovirulence to F. graminearum, which is the primary phytopathogenic fungus that causes Fusarium head blight (FHB) disease in many cereals. Understanding the interaction between mycoviruses and plant pathogenic fungi is necessary for preventing damage caused by F. graminearum. Therefore, we investigated important cellular regulatory processes in a host containing FgV1-DK21 as compared to an uninfected parent using a transcriptional approach.ResultsUsing a 3'-tiling microarray covering all known F. graminearum genes, we carried out genome-wide expression analyses of F. graminearum at two different time points. At the early point of growth of an infected strain as compared to an uninfected strain, genes associated with protein synthesis, including ribosome assembly, nucleolus, and ribosomal RNA processing, were significantly up-regulated. In addition, genes required for transcription and signal transduction, including fungal-specific transcription factors and cAMP signaling, respectively, were actively up-regulated. In contrast, genes involved in various metabolic pathways, particularly in producing carboxylic acids, aromatic amino acids, nitrogen compounds, and polyamines, showed dramatic down-regulation at the early time point. Moreover, genes associated with transport systems localizing to transmembranes were down-regulated at both time points.ConclusionThis is the first report of global change in the prominent cellular pathways in the Fusarium host containing FgV1-DK21. The significant increase in transcripts for transcription and translation machinery in fungal host cells seems to be related to virus replication. In addition, significant down-regulation of genes required for metabolism and transporting systems in a fungal host containing the virus appears to be related to the host defense mechanism and fungal virulence. Taken together, our data aid in the understanding of how FgV1-DK21 regulates the transcriptional reprogramming of F. graminearum.
Project description:To know whether the microarray technique could be used to detect bacterial gene expression in soil, large quantity of RNA was extracted from soil cultures of Pseudomonas putida KT2440 containing a chloroaromatic degrading plasmid at the presence or absence of the growth substrate, 3-chlorobenzoate (3CB). The quality and quantity of the extracted RNA were proper for a typical microarray analysis. Gene expression patterns of soil cultures were analyzed by DNA microarray using the extracted RNA. Among 5346 genes on the array, 5% and 4.5% of genes showed up- or down-regulation. Analysis done at the DAVID Bioinformatics Resources server suggested that the benzoate degradation via hydroxylation pathway had the most significant changes after treatment with 3CB. Expression of the 3CB degradation genes located in the genome was confirmed by real-time RT-PCR. In addition, real time RT-PCR analysis revealed that the fluorescent signals from plasmid genes on the microarray were saturated so that the induction ratio of the genes located in the plasmid was underestimated in microarray analysis. To our best knowledge, this report represents the first trial to use microarray technique to detect genome-wide bacterial gene expression in soil. A study using total RNA extracted from soil cultures of Pseudomonas putida KT2440/pSL1. Each chip measures the expression level of 5,341 genes from Pseudomonas putida KT2440 genome and 5 genes from an introduced plasmid pSL1 with fourteen 60-mer probes per gene which have five-fold technical redundancy.
Project description:The biocontrol agent Pseudomonas chlororaphis PA23 protects canola (Brassica napus) against infection by the necrotrophic fungus Sclerotinia sclerotiorum. Production of PA23 secondary metabolites is governed by a complex regulatory pathway that includes the GacA/GacS two component system, the PhzI/PhzR quorum-sensing system, and a novel LysR-type transcriptional regulator, called PtrA. Through RNA-sequencing, transcriptomic profiles of PA23-WT, two quorum sensing-deficient strains, PA23-AHL and PA23-phzR, and regulatory mutants PA23-gacA, PA23-gacS and PA23-ptrA were generated allowing elucidation of the PhzRI, Gac and PtrA regulons of P. chlororaphis PA23.
Project description:To gain an insight into molecular mechanisms underlying plant-microbe interactions gene expression changes in rice plants in response to a plant growth promoting rhizobacteria such as the Pseudomonas putida, root transcriptome analysis through microarray technology was performed from rice roots in response to P. putida RF3. Species of Pseudomonas are well known as biocontrol agents hence defense response and genes related to root exudation of phytochemicals were analysed in detail. For treatment of rice plants with P. putida, aseptically germinated rice seedlings from half strength MS medium were transferred to flasks containing Hoaglands’ nutrient solution, treated with P. putida and incubated for 48 hours in growth chamber in an orbital shaker. Gene expression changes in rice roots were then analyzed by microarray experiment. Untreated roots served as control. Data analysis revealed defense responsive genes to be upregulated with greater fold changes. In addition to defense response genes, few genes involved in secondary metabolism were also upregulated significantly. Validation of microarray data was performed using real time PCR for defense responsive genes (OsPBZ, OsPR101a, OsCHIA, etc). Detailed analysis of the differentially expressed genes reveal the role of P. putida RF3 in inducing systemic resistance in plants thereby immunizing the rice plants against future attacks by pests/pathogens. Our study enhances the current understanding on gene expression changes occurring during plant-microbe associations and thus demonstrates the potential of P. putida RF3 as a biocontrol agent.