Comparative genome analysis indicates high evolutionary potential of pathogenicity genes in Colletotrichum tanaceti

Colletotrichum tanaceti is an emerging foliar fungal pathogen of commercially grown pyrethrum (Tanacetum cinerariifolium). Despite being reported consistently from field surveys in Australia, the molecular basis of pathogenicity of C. tanaceti on pyrethrum is unknown. Herein, the genome of C. tanaceti (isolate BRIP57314) was assembled de novo and annotated using transcriptomic evidence. The inferred putative pathogenicity gene suite of C. tanaceti comprised a large array of genes encoding secreted effectors, proteases, CAZymes and secondary metabolites. Comparative analysis of its putative pathogenicity gene profiles with those of closely related species suggested that C. tanaceti likely has additional hosts to pyrethrum. The genome of C. tanaceti had a high repeat content and repetitive elements were located significantly closer to genes inferred to influence pathogenicity than other genes. These repeats are likely to have accelerated mutational and transposition rates in the genome, resulting in a rapid evolution of certain CAZyme families in this species. The C. tanaceti genome showed strong signals of Repeat Induced Point (RIP) mutation which likely caused its bipartite nature consisting of distinct gene-sparse, repeat and A-T rich regions. Pathogenicity genes within these RIP affected regions were likely to have a higher evolutionary rate than the rest of the genome. This “two-speed” genome phenomenon in certain Colletotrichum spp. was hypothesized to have caused the clustering of species based on the pathogenicity genes, to deviate from taxonomic relationships. The large repertoire of pathogenicity factors that potentially evolve rapidly due to the plasticity of the genome, indicated that C. tanaceti has a high evolutionary potential. Therefore, C. tanaceti poses a high-risk to the pyrethrum industry. Knowledge of the evolution and diversity of the putative pathogenicity genes will facilitate future research in disease management of C. tanaceti and other Colletotrichum spp

HRS1 Acts as a Negative Regulator of Abscisic Acid Signaling to Promote Timely Germination of Arabidopsis Seeds

In this work, we conducted functional analysis of Arabidopsis HRS1 gene in order to provide new insights into the mechanisms governing seed germination. Compared with wild type (WT) control, HRS1 knockout mutant (hrs1-1) exhibited significant germination delays on either normal medium or those supplemented with abscisic acid (ABA) or sodium chloride (NaCl), with the magnitude of the delay being substantially larger on the latter media. The hypersensitivity of hrs1-1 germination to ABA and NaCl required ABI3, ABI4 and ABI5, and was aggravated in the double mutant hrs1-1abi1-2 and triple mutant hrs1-1hab1-1abi1-2, indicating that HRS1 acts as a negative regulator of ABA signaling during seed germination. Consistent with this notion, HRS1 expression was found in the embryo axis, and was regulated both temporally and spatially, during seed germination. Further analysis showed that the delay of hrs1-1 germination under normal conditions was associated with reduction in the elongation of the cells located in the lower hypocotyl (LH) and transition zone (TZ) of embryo axis. Interestingly, the germination rate of hrs1-1 was more severely reduced by the inhibitor of cell elongation, and more significantly decreased by the suppressors of plasmalemma H+-ATPase activity, than that of WT control. The plasmalemma H+-ATPase activity in the germinating seeds of hrs1-1 was substantially lower than that exhibited by WT control, and fusicoccin, an activator of this pump, corrected the transient germination delay of hrs1-1. Together, our data suggest that HRS1 may be needed for suppressing ABA signaling in germinating embryo axis, which promotes the timely germination of Arabidopsis seeds probably by facilitating the proper function of plasmalemma H+-ATPase and the efficient elongation of LH and TZ cells

TaqMan PCR assay for detection and quantification of Stagonosporopsis tanaceti in pyrethrum seed and seedlings

Pyrethrum seed has an important role in the transmission of Stagonosporopsis tanaceti, the cause of ray blight disease of pyrethrum. A TaqMan probe based polymerase chain reaction (PCR) assay was developed to quantify the level of S. tanaceti inocula in pyrethrum seed and seedlings. Primer pair (St_qF3, St_qR2) was designed based on the intergenic spacer (IGS) region of S. tanaceti, which produced a 125 bp amplicon specific to S. tanaceti. TaqMan PCR assay using St_qF3, St_qR2 and a probe St_qP was highly specific against the genomic DNA of S. tanaceti, but did not amplify DNA of 14 related Stagonosporopsis species or other foliar pathogens of pyrethrum. The sensitivity limit of this assay was measured using the cycle threshold (Ct) value which ranged from 17.59 for 10 nanograms (ng) to 36.34 for 100 femtograms (fg) genomic DNA of S. tanaceti. There was a significant negative correlation (r = −0.999, P < 0.001) between the Ct value and the percent of S. tanaceti infected seed. In addition, this TaqMan PCR assay detected latent infection within seedlings. This assay could be applied to test commercial seed and seedlings before deciding on the appropriate management practices

Morphological and molecular characterization of Colletotrichum species from herbaceous plants in Thailand

Thirty-four isolates of Colletotrichum spp. were isolated from banana, ginger, Euphatorium thymifolia, soybean, longan, mango and Draceana sanderiana. They included endophytes from healthy plants and probable pathogens from disease lesions. Isolates were identified and grouped based on colony morphology, and size and shape of appressoria and conidia. Molecular analysis based on sequences of the rDNA internal transcribed spacers (ITS1 and ITS2), indicated that the Colletotrichum isolates comprised four clades that paralleled the morphological groupings. Most isolates clustered within three distinct clades which potentially represented different species. Endophytes isolated from different hosts are more likely to be the same species. Colletotrichum musae was positioned close to the C. gloeosporioides clades. Morphological and phylogenetic analysis of Colletotrichum pathogens and endophytes showed that endophytic isolates were most similar to C. gloeosporioides however, no pathogenic isolates clustered with endophytic isolates. The correlation between morphological and molecular-based clustering demonstrated the genetic relationships among the isolates and species of Colletotrichum and indicated that ITS rDNA sequence data were potentially useful in taxonomic species determination.link_to_OA_fulltex

Alleviation of abiotic stress in canola using genetic engineering.

Poster presentation: Biotechnology and Sustainable Futures - no. POS-THU-068ComBio2017 — the combined meeting of the Australian Society for Biochemistry and Molecular Biology, Australian Society of Plant Scientists and Australia and New Zealand Society for Cell and Developmental BiologyBrassica napus (canola) is the second largest oilseed crop grown worldwide with the annual production in Australia being around three million metric tons. The canola industry is globally affected by abiotic stress conditions such as drought, high salinity, frost and biotic stresses imposed by fungi, bacteria and insects. There is a pressing need for crop trait improvement in canola to better withstand these stress conditions. In this regard, genetic engineering could be a useful tool to introduce novel genes that have the potential to alleviate such stresses. The Acyl-CoA-Binding Protein (ACBP) family is thought to be involved in intracellular acyl-CoA ester transport and has been implicated in stress mediation in many organisms. Frost tolerant rapid-cycling B. napus and canola cv. Westar were developed by introducing the Arabidopsis thaliana ACBP6 cDNA using Agrobacterium-mediated gene transformation. Transgenic Brassica lines at T3 progeny were tested for frost tolerance in cold-acclimated and non-acclimated conditions in an upright fan-forced freezer with an electronic temperature controller. Electrolyte leakage, which arises from damage to biological membranes during freezing, was measured and found to be lower in the transgenic lines than in the wild type in non-acclimated conditions. Seed survival was higher in the transgenic lines after freezing in non-acclimated conditions. These findings indicate that the overexpression of ACBP6 is potentially useful in making canola crops more tolerant to frost in the field situations. This remains to be tested

Paraphoma chlamydocopiosa sp. nov. and Paraphoma pye sp. nov., two new species associated with leaf and crown infection of pyrethrum

Two new pathogens of pyrethrum, described as Paraphoma chlamydocopiosa and Paraphoma pye, isolated from necrotic leaf lesions on pyrethrum plants in northern Tasmania, Australia, were identified using morphological characters, phylogenetic analysis of the internal transcribed spacer (ITS), elongation factor 1-α (EF1-α) and β-tubulin (TUB) genes, and pathogenicity bioassays. Bootstrap support in the combined and individual gene region phylogenetic trees supported the two species that were significantly different from the closely related P. chrysanthemicola and P. vinacea. Morphological characteristics also supported the two new species, with conidia of P. chlamydocopiosa being considerably longer and wider than either P. chrysanthemicola or P. vinacea, and P. pye being distinct in forming bilocular pycnidia. Glasshouse pathogenicity tests based on root dip inoculation resulted in P. chlamydocopiosa and P. pye infecting the crown and upper root tissues of pyrethrum plants, and significant reduction in biomass 2 months after inoculation. Both of these Paraphoma species caused leaf lesions during in vitro and in vivo bioassays 2 weeks after foliar spray inoculation. Although P. chlamydocopiosa and P. pye were shown to be crown rot pathogens, they were also commonly isolated from leaves of diseased plants in pyrethrum fields of northern Tasmania