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

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

Subcortical brain volume, regional cortical thickness, and cortical surface area across disorders: findings from the ENIGMA ADHD, ASD, and OCD Working Groups

Objective Attention-deficit/hyperactivity disorder (ADHD), autism spectrum disorder (ASD), and obsessive-compulsive disorder (OCD) are common neurodevelopmental disorders that frequently co-occur. We aimed to directly compare all three disorders. The ENIGMA consortium is ideally positioned to investigate structural brain alterations across these disorders. Methods Structural T1-weighted whole-brain MRI of controls (n=5,827) and patients with ADHD (n=2,271), ASD (n=1,777), and OCD (n=2,323) from 151 cohorts worldwide were analyzed using standardized processing protocols. We examined subcortical volume, cortical thickness and surface area differences within a mega-analytical framework, pooling measures extracted from each cohort. Analyses were performed separately for children, adolescents, and adults using linear mixed-effects models adjusting for age, sex and site (and ICV for subcortical and surface area measures). Results We found no shared alterations among all three disorders, while shared alterations between any two disorders did not survive multiple comparisons correction. Children with ADHD compared to those with OCD had smaller hippocampal volumes, possibly influenced by IQ. Children and adolescents with ADHD also had smaller ICV than controls and those with OCD or ASD. Adults with ASD showed thicker frontal cortices compared to adult controls and other clinical groups. No OCD-specific alterations across different age-groups and surface area alterations among all disorders in childhood and adulthood were observed. Conclusion Our findings suggest robust but subtle alterations across different age-groups among ADHD, ASD, and OCD. ADHD-specific ICV and hippocampal alterations in children and adolescents, and ASD-specific cortical thickness alterations in the frontal cortex in adults support previous work emphasizing neurodevelopmental alterations in these disorders

Asparagus virus 2 : incidence, yield effects and transmission in New Zealand asparagus crops

Asparagus ilarvirus 2 (AV2) occurs in asparagus crops in New Zealand and worldwide, but its effect on and spread within crops has not been reported in the literature. The causes of declining yields in asparagus crops, reported in recent years, are also not well understood. This investigation examined the incidence of AV2 within New Zealand, its effects on yield and its spread within asparagus crops. By using an indirect ELISA method, that was developed for detection of the virus, AV2 was shown to be present in 93% of the 67 New Zealand asparagus fields surveyed and in 44% of sampled plants. Incidence was higher in older plantings, indicating that secondary spread of the virus had occurred. A field trial with clonal plants showed that, for the years 1992-1996, AV2 infection caused annual reductions in mean marketable spear yields of 14%, 28%, 20%, 48% and 57% and increases in mean reject spear yields of 93%, 105%, 207%, 352% and 167%, respectively. Spear yields from AV2-free plants increased annually over the life of the trial and although yields from AV2-infected plants increased to the third year of harvest, they decreased thereafter. In harvest years four and five, AV2- infected plants produced fewer, smaller spears and fern stalks than AV2-free plants. Transmission of AV2 by cutting knives, was investigated in a separate field trial in which the AV2-infected plants were placed in the middle of rows with AV2-free plants on either side. During 1992-1995, spears were harvested by cutting each row in one direction only. One block of 12 rows was cut in the same direction as the prevailing wind and the other block in the opposite direction. Plants were tested for AV2 annually. By 1995, AV2 had spread into 96% of plants cut after the AV2 source plants, indicating transmission by cutting, and into 26% of plants cut prior to the source plants, predominantly into female plants and those downwind of the source plants, indicating wind-assisted transmission. The parental source of AV2 in infected seed was investigated by hand crosspollination between AV2-infected and AV2-free parent plants of each gender, growing in an insect-proof greenhouse. ELISA tests of shoots from the resulting seedlings showed that when only one parent was infected, AV2 was transmitted into 17% and 47% of seedlings, from male and female parents respectively, and into 95% of seedlings when both parents were infected. Infection of parent plants resulted in reductions in successful pollination, size and number of seed, germination and seedling viability. Of the adult plants originally free from AV2, which had been pollinated with AV2-infected pollen, 83% tested positive for AV2 the following year, indicating transmission of AV2 during pollination. Because the same symptoms shown by AV2-infected plants have been reported as being characteristic of asparagus decline syndrome, in which asparagus crops become progressively less productive and are often uneconomic by the tenth harvest, AV2 must be considered to be a contributing factor to asparagus decline

Development of a PCR-RFLP method to distinguish species within the Ilyonectria macrodidyma complex

Species within the Ilyonectria macrodidyma complex are known plant pathogens and several are implicated as the causal agents of black foot disease of grapevines. The seven species within the complex can be identified by DNA sequencing of the histone H3 gene. In this study, a PCR-RFLP method to identify the species was developed. In silico digestion of the 500 bp histone H3 amplimer using MnlI showed that it could identify Ilyonectria sp. 1, Ilyonectria sp. 2, I. alcacerensis and I. macrodidyma. Subsequent in silico digestion with Hinf1 identified I. estremocensis, I. novozelandica and I. torresensis. The PCR-RFLP was validated using a collection of 40 I. macrodidyma complex isolates that had been recovered from symptomatic grapevines. Ilyonectria macrodidyma, I. novozelandica and I. torresensis were detected in that collection. Intraspecific polymorphism was only detected in I. torresensis. This method provides a rapid procedure for identifying individual species of the I. macrodidyma species complex

Distribution of Botryosphaeriaceae species and genotypes within a rootstock mother vine indicates multiple inoculum sources

Infected rootstock and scion cuttings by Botryosphaeriaceae fungi have been reported as major sources of infection for young grapevines. To investigate the potential infection pathways of Botryosphaeriaceae species within a rootstock mother vine, a universally-primed polymerase chain reaction (UP-PCR) was conducted. This method differentiated the genotypes within two Neofusicoccum species, namely N. luteum and N. parvum. The first study showed that multiple Botryosphaeriaceae species and genotypes can infect a single mother vine. It further showed that the trunk and shoot isolates of the same species from the same vine were of the same or different genotypes, suggesting multiple infections from different inoculum sources. The second study that investigated the spatial distribution of Botryosphaeriaceae fungi within an entire dormant cane also showed that multiple species and genotypes were distributed along the cane, but most isolates were sited within the bark and less frequently in the wood, suggesting they were latent in surface tissues. Some adjacent wood and bark infections were caused by the same genotypes also suggesting that wood infection may have originated from the bark. The third study further showed that the Neofusicoccum isolates recovered by washing the cuttings were of the same or different genotypes from those isolated from adjacent internal tissues, again suggesting multiple sources of external inoculum. These fungi appear to cause latent infections in the bark of dormant cuttings which are used in plant propagation, thus providing an additional infection pathway for a disease that is known to show obvious symptoms only in older vineyards

Co-expression of two genes, a chitinase (chit42) and proteinase (prb1), implicated in mycoparasitism by Trichoderma hamatum

Mycoparasitism of fungal plant pathogens by Trichoderma species is a complex process that involves the production and coordinated secretion of cell-wall degrading enzymes. Genes implicated in mycoparasitism by Trichoderma atroviride contain motifs in the promoter region, designated MYRE1-MYRE4, that are proposed to act as binding sites for a global inducer of the mycoparasitic response. The aim of our study was to establish whether these motifs also were present in Trichoderma hamatum and whether the presence of these motifs could predict co-expression when T. hamatum was confronted by a pathogen. Using a combination of targeted, degenerate and inverse PCR, homologues of the mycoparasitism-related genes ech42 (chit42), prb1 and lam1.3 (xbg1.3-110), which encode an endochitinase, proteinase, and β-1,3-glucanase, respectively, were cloned and sequenced from T. hamatum. Alignment of the promoter regions of the three genes revealed identical regions in the chit42 and prb1 promoters, which were 6–9 base pairs in length and conserved in position. Specifically, the regulator y motifs MYRE1-MYRE4 were fully conserved, together with a fifth motif, identified by this research. A substrate assay designed to investigate the response of these genes from T. harzianum and T. hamatum to a simple carbon source (glycerol) showed that, in contrast to chit42 and prb1, xbg1.3-110 was not expressed. Further comparison of the expression patterns of these three genes between T. harzianum and T. hamatum using the glycerol substrate assay showed that no chit42 or prb1 expression could be detected in T. harzianum when it was grown under the same conditions as T. hamatum. This showed that the response of these genes to glycerol was species specific and that a single expression pattern for these genes was not common to all Trichoderma species. Confrontation assays were used to investigate the response of the three T. hamatum genes to the more complex substrate posed by the fungal pathogen Sclerotinia sclerotiorum. Once again gene expression analysis showed that both chit42 and prb1 were co-expressed and moderately induced during confrontation against Sclerotinia sclerotiorum. Although xbg1.3-110 previously had been implicated in mycoparasitism by T. harzianum, this study detected no xbg1.3-110 expression during confrontation between T. hamatum and S. sclerotiorum. These findings show that the MYRE1-MYRE4 together with MYRE5 are present in two species of Trichoderma, T. atroviride and T. hamatum and that the presence of these motifs could predict co-expression in response to two carbon sources

Susceptibility of four grapevine rootstocks to Cylindrocladiella parva

The susceptibility of four common grapevine rootstocks (101-14, Schwarzmann, 5C and Riparia Gloire) to Cylindrocladiella parva (black foot disease) infection was assessed in a pot experiment. The roots of 4-month-old callused rooted cuttings were wounded in situ and inoculated with 50 ml C. parva conidial suspension (106/ml) or sterile water (controls). After 6 months of growth, shoot dry weight was significantly higher for control plants (24.2 g) than for those inoculated with C. parva (16.5 g), but did not differ between rootstock varieties. Root dry weight was not significantly affected by C. parva inoculation, but root dry weight of 101-14 was significantly higher than other rootstocks. Incidence and severity of trunk infection were significantly affected by rootstock variety, being lowest in rootstock 101-14 plants than other rootstocks. None of the rootstocks tested was resistant to this pathogen