Hayley J. Ridgway

Approaches to functional genomics in filamentous fungi.

The study of gene function in filamentous fungi is a field of research that has made great advances in very recent years. A number of transformation and gene manipulation strategies have been developed and applied to a diverse and rapidly expanding list of economically important filamentous fungi and oomycetes. With the significant number of fungal genomes now sequenced or being sequenced, functional genomics promises to uncover a great deal of new information in coming years. This review discusses recent advances that have been made in examining gene function in filamentous fungi and describes the advantages and limitations of the different approaches.

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.

Molecular evidence for the presence of Phaeomoniella chlamydospora in New Zealand nurseries, and its detection in rootstock mothervines using species-specific PCR

Phaeomoniella chlamydospora is considered to be the causal agent of Petri disease of grapevines. The source of primary inoculum is believed to be through the planting of grafted vines collected from infected mother rootstock vines. The presence of P. chlamydospora in New Zealand has been confirmed by analysis of the ribosomal gene region amplified from morphologically identified isolates. Sequencing of six of these isolates revealed that their nucleotide sequences were 99.9–100% similar to those reported in GenBank, and established that the species-specific primers, previously developed for overseas strains, would amplify the New Zealand isolates. A method for DNA extraction from grapevine wood was developed which, when combined with the species-specific primers, provides the basis for a diagnostic service for the detection of this disease in rootstock.

Identification, potential inoculum sources and pathogenicity of botryosphaeriaceous species associated with grapevine dieback disease in New Zealand

This study investigated the prevalence and identity of botryosphaeriaceous dieback pathogens in necrotic grapevines tissues in New Zealand vineyards, and other woody hosts growing nearby. The presumptive identities of the isolates by conidial and cultural morphology were confirmed with ITS sequence data as Neofusicoccum australe, N. luteum, N. parvum and Diplodia seriata. They were isolated predominantly from necrotic stems of grapevine and other hosts, but also from leaves, flowers and wood debris of grapevines. Inoculation with conidia and mycelium of multiple isolates of each species onto excised and attached green shoots and trunks of five grapevine varieties, Cabernet sauvignon, Chardonnay, Pinot noir, Riesling, and Sauvignon blanc, showed that all varieties became infected to a similar extent. All species except D. seriata were pathogenic, irrespective of the host source, with N. luteum being the most and D. mutila the least pathogenic (P < 0.05). On trunks, N. parvum caused cankers and the other pathogenic species caused die-back when the inoculated vines became winter-dormant. Conidia were produced from green shoot lesions and die-back wood, which indicates potential inoculum sources for vineyard infection.

Genetic basis of mycoparasitism: A mechanism of biological control by species of Trichoderma

Abstract Biocontrol of fungal plant pathogens through the use of mycoparasitic fungi is an environmentally sustainable approach to management of plant diseases. Mycoparasitism by Trichoderma spp. primarily involves production of cell‐wall degrading enzymes. Isolation and characterisation of the corresponding genes have revealed major insights into the underlying genetic basis of mycoparasitism, implicating various regulatory pathways such as carbon and nitrogen catabolite repression. A summary is presented here of the current state of knowledge in molecular regulation of mycoparasitism by Trichoderma species.

Burkholderia sp. Induces Functional Nodules on the South African Invasive Legume "Dipogon lignosus" (Phaseoleae) in New Zealand Soils

The South African invasive legume Dipogon lignosus (Phaseoleae) produces nodules with both determinate and indeterminate characteristics in New Zealand (NZ) soils. Ten bacterial isolates produced functional nodules on D. lignosus. The 16S ribosomal RNA (rRNA) gene sequences identified one isolate as Bradyrhizobium sp., one isolate as Rhizobium sp. and eight isolates as Burkholderia sp. The Bradyrhizobium sp. and Rhizobium sp. 16S rRNA sequences were identical to those of strains previously isolated from crop plants and may have originated from inocula used on crops. Both 16S rRNA and DNA recombinase A (recA) gene sequences placed the eight Burkholderia isolates separate from previously described Burkholderia rhizobial species. However, the isolates showed a very close relationship to Burkholderia rhizobial strains isolated from South African plants with respect to their nitrogenase iron protein (nifH), N-acyltransferase nodulation protein A (nodA) and N-acetylglucosaminyl transferase nodulation protein C (nodC) gene sequences. Gene sequences and enterobacterial repetitive intergenic consensus (ERIC) PCR and repetitive element palindromic PCR (rep-PCR) banding patterns indicated that the eight Burkholderia isolates separated into five clones of one strain and three of another. One strain was tested and shown to produce functional nodules on a range of South African plants previously reported to be nodulated by Burkholderia tuberum STM678T which was isolated from the Cape Region. Thus, evidence is strong that the Burkholderia strains isolated here originated in South Africa and were somehow transported with the plants from their native habitat to NZ. It is possible that the strains are of a new species capable of nodulating legumes.

Incidence and distribution of botryosphaeriaceous species in New Zealand vineyards

The aim of this study was to determine the identification, incidence and distribution of botryosphaeriaceous species in New Zealand vineyards. A field study of 43 vineyards across six wine growing regions was conducted. A total of 336 isolates of botryosphaeriaceous species were isolated from 238 diseased grapevine samples. Morphological identification and phylogenetic analysis of the ribosomal RNA gene region, partial sequence of the translation elongation factor 1-alpha gene (EF1-α) and the β-tubulin gene identified nine botryosphaeriaceous species: N. parvum, N. luteum, N. australe, N. ribis, D. mutila, D. seriata, B. dothidea, Do. iberica and Do. sarmentorum. These species have been reported in other grape growing regions worldwide. Eighty eight percent of vineyards and 68% of symptomatic vines sampled were positive for botryosphaeriaceous species. Incidence and distribution of the botryosphaeriaceous species populations varied between the North and South Islands with N. parvum being the predominant species. The variability in incidence and distribution of the botryosphaeriaceous species may be influenced by climatic conditions and different sources of inoculum in the regions sampled. The results of this research clearly identified botryosphaeriaceous species as the target pathogens for development of management strategies for grapevine decline in New Zealand.

Evaluation of fungicides for the management of Botryosphaeria dieback diseases of grapevines.

BACKGROUND A range of botryosphaeriaceous species can cause dieback and cankers in grapevines; however, different species most commonly affect the grapevines in different grape-growing regions and countries. They infect through wounds and sporulate on woody stems and green shoots throughout the year, so wound protection is the recommended control strategy. This research evaluated fungicides for their ability to reduce mycelial growth and conidial germination of three botryosphaeriaceous species and to protect pruning wounds against infection. RESULTS In vitro experiments showed that nine out of 16 tested fungicides were effective at reducing mycelial growth and/or conidial germination of three isolates each of Neofusicoccum australe, N. luteum and Diplodia mutila. The species differed in their response to the fungicides, although N. luteum was usually the least sensitive. When nine selected fungicides were sprayed on cane pruning wounds on potted and field grapevines and subsequently inoculated with N. luteum conidia, some effectively protected them from infection. The most effective fungicides were flusilazole, carbendazim, tebuconazole, thiophanate-methyl and mancozeb, as they prevented the inoculated pathogen from infecting healthy wood in 100, 93, 87, 83 and 80% of field vines, respectively. CONCLUSION This research has demonstrated that fungicides applied after winter pruning can protect vines from infection by conidia of three botryosphaeriaceous species.

Infection of rootstock mother-vines by Phaeomoniella chlamydospora results in infected young grapevines

The use of cutting material infected with Phaeomoniella chlamydospora is believed to contribute to Petri disease. Infection levels of own-rooted scion, own-rooted rootstock and grafted vines were compared in a 20-month field trial. In own-rooted rootstock and grafted vines, infection increased over time (53% and 20%, respectively, at the 20-month assessment). Infection was not observed in own-rooted scion until the 20-month assessment (22%). It was concluded that rootstock mother-vines are a primary source of the pathogen. The ability of the pathogen to move from the retained rootstock mother-vine trunk or head, into current season’s growth was investigated by determining disease levels in own-rooted rootstock vines propagated from cuttings collected at different distances from the head. Vines from dormant cuttings collected nearest the head had a higher level of infection at the 8-month assessment (42%) than those collected from further away (6–8%). At the 20-month assessment, infection in vines from close to the head and vines from actively growing cuttings collected furthest from the head were similar (53 and 55%, respectively). Black streaking, a commonly used disease assessment criterion,was found to overestimate disease. Possible sources of inoculum and disease management options for nurseries are discussed.

Genetic and pathogenic diversity of Neofusicoccum parvum in New Zealand vineyards

Genetic diversity of 50 isolates of Neofusicoccum parvum, the predominant species of the Botryosphaeriaceae recovered from grapevines displaying symptoms of dieback and decline in New Zealand, was compared to that of isolates from Australia, South Africa, and California. The eight universally primed polymerase chain reaction (UP-PCR) primers distinguished 56 genotypes, with only four clonal pairs found. Seven main groups were identified in a neighbour-joining (NJ) tree with isolates from different regions and vineyards of New Zealand, Australia, and California distributed in different groups, indicating a high level of intra and intervineyard genetic variation. All of the South African isolates were positioned in a separate UP-PCR group, indicating that these isolates were less related to the other N. parvum isolates. When compared to fungi that reproduce sexually the genetic diversity and Shannon diversity indices were low (0.076-0.249; 0.109-0.367, respectively), genetic identity levels were high (0.76-0.95), and genetic distance levels were low (0.04-0.27). The large number of genotypes and the low number of clones in the New Zealand N. parvum populations may be explained by parasexual recombination as anastomosis was observed between nonself pairings. Pathogenicity tests using isolates from different UP-PCR groups inoculated onto either green shoots or 1-y-old grapevines detected virulence diversity, indicating intra and intervineyard variation between isolates, however, no correlation was detected between UP-PCR group and virulence.