Genetic diversity and virulence variability in Diplodia mutila isolates from symptomatic grapevines in New Zealand: Virulence and genetic diversity of Diplodia mutila

Genetic diversity and virulence variability of Diplodia mutila isolates recovered from grapevines in New Zealand were investigated. The universally primed PCR (UP-PCR) and vegetative compatibility group (VCG) methods were used to investigate the genetic diversity. Pathogenicity tests with ‘Sauvignon Blanc’ detached shoots and potted vines were used to determine the virulence diversity. UP-PCR analysis determined eight genetic groups of D. mutila with 70% of the population within one group. Phylogenetic analysis also determined that New Zealand isolates were more closely related to Australian isolates than Californian isolates. Vegetative compatibility grouping analysis placed the isolates into three VCG groups, with 57% of isolates belonging to all three VCGs. Vegetative compatibility reactions were observed among isolates, but this was not correlated with the genetic clustering. Virulence assays proved that all isolates tested were pathogenic on grapevine stems. Differences in necrotic lesions lengths caused by D. mutila isolates were identified, indicating different virulence levels among isolates, however, no relationship was found between the genetic groups and the virulence. The results of the study indicated movement of D. mutila isolates between nurseries, vineyards, and other sources in New Zealand. This information will inform control strategies to limit the further spread of this pathogen into vineyards in the same region or new regions

Determining the presence of host specific toxin genes, ToxA and ToxB, in New Zealand Pyrenophora tritici-repentis isolates, and susceptibility of wheat cultivars

Tan spot, caused by Pyrenophora tritici-repentis (Ptr), is an important disease of wheat worldwide, and an emerging issue in New Zealand. The pathogen produces host-specific toxins which interact with the wheat host sensitivity loci. Identification of the prevalence of the toxin encoding genes in the local population, and the susceptibility of commonly grown wheat cultivars to Ptr will aid selection of wheat cultivars to reduce disease risk. Twelve single spore isolates collected from wheat-growing areas of the South Island of New Zealand representing the P. tritici-repentis population were characterised for the Ptr ToxA and ToxB genes, ToxA and ToxB, respectively, using two gene specific primers. The susceptibility of 10 wheat cultivars to P. tritici-repentis was determined in a glasshouse experiment by inoculating young plants with a mixed-isolate spore inoculum. All 12 New Zealand P. tritici-repentis isolates were positive for the ToxA gene but none were positive for the ToxB gene. Tan spot lesions developed on all inoculated 10 wheat cultivars, with cultivars ‘Empress’ and ‘Duchess’ being the least susceptible and ‘Discovery’, ‘Reliance’ and ‘Saracen’ the most susceptible cultivars to infection by the mixed-isolate spore inoculum used. The results indicated that the cultivars ‘Empress’ and ‘Duchess’ may possess a level of tolerance to P. tritici-repentis and would, therefore, be recommended for cultivation in regions with high tan spot incidence

Similar strains of Burkholderia spp. nodulate the South African invasive legume Dipogon lignosus in New Zealand and Australian soils

Brazil and South Africa are centres of diversity of Burkholderia ssp. that nodulate legumes (Gyaneschwar et al, 2011; Beukes et al. 2013). The nod gene sequence of Burkholderia spp, capable of nodulating South Africa plants are clearly separated from those of Burkholderia spp. shown to nodulate South American plants. Where tested, the South African strains did not nodulate South American plants nodulated by Burkholderia spp. (Gyaneschwar et al. 2011)

Grapevines escaping trunk diseases in New Zealand vineyards have a distinct microbiome structure

Grapevine trunk diseases (GTDs) are a substantial challenge to viticulture, especially with a lack of available control measures. The lack of approved fungicides necessitates the exploration of alternative controls. One promising approach is the investigation of disease escape plants, which remain healthy under high disease pressure, likely due to their microbiome function. This study explored the microbiome of grapevines with the disease escape phenotype. DNA metabarcoding of the ribosomal internal transcribed spacer 1 (ITS1) and 16S ribosomal RNA gene was applied to trunk tissues of GTD escape and adjacent diseased vines. Our findings showed that the GTD escape vines had a significantly different microbiome compared with diseased vines. The GTD escape vines consistently harbored a higher relative abundance of the bacterial taxa Pseudomonas and Hymenobacter. Among fungi, Aureobasidium and Rhodotorula were differentially associated with GTD escape vines, while the GTD pathogen, Eutypa, was associated with the diseased vines. This is the first report of the link between the GTD escape phenotype and the grapevine microbiome

The relative susceptibility of grapevine rootstocks to black foot disease is dependent on inoculum pressure

Black foot disease of grapevines is a major economic issue for the viticulture industry worldwide. The disease is mainly associated with a complex of pathogen species within the genera Dactylonectria and Ilyonectria. The susceptibility of six grapevine rootstock cultivars to black foot disease under field conditions was assessed. Callused rootstocks of 101-14, 5C, 420A, Riparia Gloire, Schwarzmann and 3309C were planted into soil containing low natural pathogen populations or inoculated with isolates representing the species diversity in New Zealand. Disease incidence, disease severity and dry weight accumulation were assessed after 8 months of growth. Root and shoot dry weights were not significantly affected by inoculation treatment, but differed among rootstock cultivars, with cultivar 420A having the lowest root and shoot dry weight, cultivar 3309C having the largest shoot dry weight and cultivar 5C the largest root dry weight. The relative susceptibility of rootstocks differed significantly depending on whether they were grown under low natural inoculum pressure or a higher pressure in artificially inoculated soil. Schwarzmann and Riparia Gloire rootstock cultivars were the least susceptible under natural low inoculum pressure, but were the most susceptible in inoculated soil. In contrast, 5C was one of the most susceptible under low inoculum levels but was the least susceptible under high pathogen pressure. The result of the study indicate that black foot pathogen inoculum levels in soil affect the relative susceptibility of grapevine rootstocks to infection, and may have implications for the selection of rootstocks for planting

The identity, distribution and diversity of botryosphaeriaceous species in New Zealand vineyards – a national perspective

In recent years molecular tools have been applied to provide understanding of the population structures of botryosphaeriaceous species in New Zealand vineyards. A national survey of symptomatic material from 43 vineyards showed that 88% had infection by botryosphaeriaceous species. Vine age had the strongest correlation with incidence, with the least infection in grapevines 1–5 years old (30%). Sequencing of taxonomic genes identified nine species. In contrast to other countries, N. luteum and N. parvum were predominant species with Lasiodiplodia theobromae notably absent. As with other countries, research showed that distribution is likely to be related to climate. Analysis of populations demonstrated that, despite predominantly asexual reproduction, the genetic diversity of isolates within species was high. Frequent hyphal anastomoses and fusions were observed in dual culture with weak vegetative compatibility barriers. This indicated the likelihood of frequent parasexual recombination. The isolation of genetically similar isolates from single lesions reinforced this hypothesis. A suite of molecular tools were developed to aid epidemiology studies. Endogenous markers produced for isolates with typical pathogenesis showed they could be dispersed at least 2 m from the site of conidiation in a single rain/wind event. The use of a multi-genus PCR-SCP system showed that N. parvum and N. luteum are released year round and this probably contributes to their successful invasion of vineyards. Application of these molecular tools has provided a comprehensive snapshot of New Zealand vineyards revealing a thriving and diverse population of botryosphaeriaceous species that present a serious concern to the industry

Pathway level subtyping identifies a slow-cycling biological phenotype associated with poor clinical outcomes in colorectal cancer

Molecular stratification using gene-level transcriptional data has identified subtypes with distinctive genotypic and phenotypic traits, as exemplified by the consensus molecular subtypes (CMS) in colorectal cancer (CRC). Here, rather than gene-level data, we make use of gene ontology and biological activation state information for initial molecular class discovery. In doing so, we defined three pathway-derived subtypes (PDS) in CRC: PDS1 tumors, which are canonical/LGR5+ stem-rich, highly proliferative and display good prognosis; PDS2 tumors, which are regenerative/ANXA1+ stem-rich, with elevated stromal and immune tumor microenvironmental lineages; and PDS3 tumors, which represent a previously overlooked slow-cycling subset of tumors within CMS2 with reduced stem populations and increased differentiated lineages, particularly enterocytes and enteroendocrine cells, yet display the worst prognosis in locally advanced disease. These PDS3 phenotypic traits are evident across numerous bulk and single-cell datasets, and demark a series of subtle biological states that are currently under-represented in pre-clinical models and are not identified using existing subtyping classifiers

Epithelial TGFβ engages growth-factor signalling to circumvent apoptosis and drive intestinal tumourigenesis with aggressive features

The pro-tumourigenic role of epithelial TGFβ signalling in colorectal cancer (CRC) is controversial. Here, we identify a cohort of born to be bad early-stage (T1) colorectal tumours, with aggressive features and a propensity to disseminate early, that are characterised by high epithelial cell-intrinsic TGFβ signalling. In the presence of concurrent Apc and Kras mutations, activation of epithelial TGFβ signalling rampantly accelerates tumourigenesis and share transcriptional signatures with those of the born to be bad T1 human tumours and predicts recurrence in stage II CRC. Mechanistically, epithelial TGFβ signalling induces a growth-promoting EGFR-signalling module that synergises with mutant APC and KRAS to drive MAPK signalling that re-sensitise tumour cells to MEK and/or EGFR inhibitors. Together, we identify epithelial TGFβ signalling both as a determinant of early dissemination and a potential therapeutic vulnerability of CRC’s with born to be bad traits

Pathway level subtyping identifies a slow-cycling biological phenotype associated with poor clinical outcomes in colorectal cancer

Molecular stratification using gene-level transcriptional data has identified subtypes with distinctive genotypic and phenotypic traits, as exemplified by the consensus molecular subtypes (CMS) in colorectal cancer (CRC). Here, rather than gene-level data, we make use of gene ontology and biological activation state information for initial molecular class discovery. In doing so, we defined three pathway-derived subtypes (PDS) in CRC: PDS1 tumors, which are canonical/LGR5+ stem-rich, highly proliferative and display good prognosis; PDS2 tumors, which are regenerative/ANXA1+ stem-rich, with elevated stromal and immune tumor microenvironmental lineages; and PDS3 tumors, which represent a previously overlooked slow-cycling subset of tumors within CMS2 with reduced stem populations and increased differentiated lineages, particularly enterocytes and enteroendocrine cells, yet display the worst prognosis in locally advanced disease. These PDS3 phenotypic traits are evident across numerous bulk and single-cell datasets, and demark a series of subtle biological states that are currently under-represented in pre-clinical models and are not identified using existing subtyping classifiers

Investigation of a number of dysfibrinogenaemias and their impact on fibrinogen function

An understanding of fibrinogen activation and polymer formation has been derived from the study of natural fibrinogen variants (dysfibrinogenaemias). This thesis details the investigation of a number of dysfibrinogenaemias and how they impact on fibrinogen function. Fibrinogen Lincoln results from translation of an Aα chain gene containing a 13 bp deletion (nt 4758-4770), which causes a frameshift and translates as four new amino acids before termination at a stop codon. Turbidity curves of fibrin monomers revealed that polymerisation was delayed and the final turbidity was half that of the control. Decreased final turbidity suggested that the fibrin fibres formed were of a thinner diameter than normal. Scanning electron micrographs supported these findings revealing a clot network composed of thinner fibres and smaller pores. The decreased pore size of the fibrin network was substantiated by permeation data, which revealed that clots were 25 fold less permeable. Fibrinogen Otago was found in a woman diagnosed with afibrinogenaemia. She was later found to have a functional fibrinogen concentration of 0.06 mg/ml. Plasma protein electrophoresis showed an absence of normal fibrinogen and a novel 270 kDa component, which was confirmed as fibrinogen by immunofixation. Reducing SDS-PAGE showed that the expected 67 kDa Aα chain was missing and replaced by a 30 kDa band. This aberrant chain was not detected by the monoclonal antibody F-103. Cycle sequencing of the DNA encoding the F-103 epitope revealed the homozygous insertion of cytosine within a block of four cytosines at position 4133 bp of the Aα gene sequence. The new sequence translates with three new amino acids before termination at a stop codon. Turbidity studies of fibrin monomers revealed delayed polymerisation, with a final turbidity higher than the control. Although this suggested that the final fibre diameter was larger than normal, scanning electron micrographs revealed clots composed of thinner fibres and smaller pores. Fibrinogen Kaiserslautern was found in an adult woman with prolonged thrombin and reptilase times. Analysis of five other family members also revealed prolonged, but variable, thrombin and reptilase times. Reducing SDS-PAGE revealed an additional protein band, in all family members, which migrated immediately below the normal Bβ band. Western blotting confirmed that this band was a γ chain and endoglycosidase-F digestion established that it contained an additional oligosaccharide. Partial acid digestion defined the location of the new oligosaccharide to the C-terminus of the γ chain. Cycle sequencing of this region revealed a single base substitution altering the normal Lys (AAG) codon to Asn (AAT), producing a new Asn-X-Thr glycosylation site. The patient and one other family member were homozygous for this mutation while the remaining four family members were heterozygous. The polymerisation of fibrin monomers from the patient was abnormal, however, it was normalised by the removal of sialic acid residues with neuraminidase. This suggested that the polymerisation defect was primarily caused by negatively charged sialic acid residues present on the oligosaccharide. Further analysis of the D domain of purified fibrinogen established that calcium binding to the high affinity site was unaffected by the oligosaccharide side chain or negatively charged sialic acid residues. Factor XIIIa catalysed crosslinking of the fibrinogen was delayed whereas the crosslinking of fibrin was normal. Scanning electron micrographs of plasma from the patient revealed clots which were similar to the control. A number of mutations were detected in the N-terminal of the Aα chain. The Aα 16 Arg to His mutation was characterised using PCR and restriction digestion. The mutation in Fibrinogen Oslo IV (Aα 19 Arg→Gly) was found by DNA sequencing of exon II of the Aα chain gene and confirmed by N-terminal protein sequencing. This is the third reported case of this mutation. Protein sequencing of fibrin from Otago II detected two amino acid sequences. The first sequence was the normal Gly¹⁷-Pro-Arg-Val-Val... and the second was Asp²⁰-Val-Glu-Arg ... This suggested that the patient was heterozygous for a point mutation, which alters Aα 20 valine to aspartate. This mutation creates a new proprotein processing site. These three variants illustrate different mechanisms by which point mutations can affect protein structure and function